KR20140096029A - Methods for treating hcv - Google Patents

Abstract

The present invention relates to a combination of therapeutic molecules useful for treating hepatitis C virus infection. The present invention relates to methods, uses, administration regimens, and compositions.

Description

{METHODS FOR TREATING HCV}

Priority of invention

This application is related to U.S. Provisional Application No. 61 / 535,885, filed September 16, 2011; And U.S. Provisional Application No. 61 / 561,753, filed November 18, 2011. The entire contents of each of which is incorporated herein by reference.

Field of invention

The present invention relates to a combination of therapeutic molecules useful for treating hepatitis C virus infection. The present invention relates to methods, uses, dosing regimens and compositions.

Hepatitis is a worldwide disease. When considering the state of chronic inflammation in the liver, hepatitis is generally caused by viruses, although there may be other known non-infectious causes. Viral hepatitis is by far the most common form of hepatitis. The U.S. Centers for Disease Control estimates that more than 1.8% of the US population has serological evidence of HCV infection in a number of cases involving chronic active infections. HCV is a positive-strand RNA virus belonging to Flaviviridae and is closely related to pestiviruses including porcine cholera virus and bovine viral diarrhea virus.

The HCV genome is a single-stranded, positive directional RNA of about 9,600 bp encoding a multi-protein of 3009-3030 amino acids, and is a mature viral protein after translation and translation and translation by the cell and two viral proteases , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). The structural proteins E1 and E2 are known to be embedded in a viral lipid envelope to form a stable heterodimer. Structural core proteins are known to interact with viral RNA genomes to form nucleocapsids. Nonstructural proteins designated NS2 through NS5 include proteins with enzymatic action associated with viral replication and protein processing, including polymerases, proteases and helicases. HCV replicates through the generation of complementary negative-strand RNA templates.

HCV is a genetically diverse virus. In single infected patients, many variant viruses or viral species can be identified in accordance with the description of the 'viral flock'. In the world population, HCV is also genetically diverse, with more than six major 'genotypes' identified (genotypes 1-6) and many subtypes (ie, HCV genotypes 1a and 1b). HCV genotypes are defined by genomic phylogenetic analysis and diagnosed (in a given patient) by HCV RNA sequence-based diagnostic assays.

The main route of infection with HCV is bleeding. The severity of HCV infection as a health problem is explained by the prevalence among high risk groups. For example, some studies show that 60% to 90% of hemophiliacs in the West and over 80% of intravenous drug addicts have chronic HCV infection. For intravenous drug addicts, the prevalence varies from about 28% to 80%, depending on the studied population. The proportion of new HCV infections associated with transfusion or blood product input was significantly reduced due to pharmacologic advances and widespread use of sensitive serology and RNA detection assays used to screen blood donors, but a large group of aging, chronic Infected people are already known.

One possible treatment for HCV infection is pegylated interferon-alpha (PEG-IFNa1a or PEG-IFNa1b) and is administered weekly by subcutaneous injection for 24-48 weeks, depending on the HCV viral genotype to be treated, under current treatment guidelines . Although more than 50% of patients with genotype 1 HCV infection can expect inhibition of HCV viremia at the completion of 48 weeks of therapy, a significant proportion of these patients will have a viral recurrence. Thus, a sustained viral response (SVR, defined as HCV RNA negative at 24 weeks after discontinuation of treatment and considered as "treatment") is only 30-40% of the genotype 1 HCV infection treated with PEG-IFN alone . In addition, treatment with PEG-IFN + RBV is not resistant to adverse effects profiles including cold-like symptoms, thrombocytopenia, anemia and severe psychiatric side effects. While treatment to the current standard of care is second-lane, many patients are hampered by start-up therapy due to common comorbidities in the HCV-infected population, including psychiatric disorders, severe liver disease, and drug abuse.

Ribavirin is a nucleoside-like antiviral drug. Ribavirin is usually taken orally twice a day. The exact mechanism of ribavirin is not known. However, when ribavirin enters a cell, it is phosphorylated; It is then known to act as an inhibitor of inosine 5'-monophosphate dehydrogenase (IMPDH). IMPDH inhibitors, such as ribavirin, inhibit viral replication by reducing the nucleotide "building blocks" required for intracellular synthesis and storage of guanine, DNA and RNA production. IMPDH inhibitors also interfere with the reproduction of rapidly proliferating cells and cells with a high rate of protein transduction. Treatment with ribavirin monotherapy has little effect on HCV RNA levels, but is associated with a decrease in serum alanine transferase (ALT). This observation suggests that ribavirin will act as a mediator of immune system function rather than acting as an antiviral agent. Ribavirin is only approved for use in combination with IFN in connection with HCV infection.

Due to the reduced frequency of viral recurrence at the discontinuation of therapy, treatment of the PEG-IFN + ribavirin combination greatly improves the SVR ratio compared to the treatment observed with PEG-IFN alone. The SVR ratio for large-scale clinical trials for patients with HCV genotype 1 infection treated with PEG-IFN / ribavirin ranges from 40-55%. Currently, PEG-IFN / ribavirin therapy is considered a 'standard of care' treatment for chronic HCV infection. However, the standard of care is expected to change rapidly in the near future with the approval of direct acting antiviral agents, which will be used for the first time in combination with PEG-IFN / ribavirin.

Unfortunately, different HCV genotypes respond differently to PEG-IFN / ribavirin therapy; For example, HCV genotype 1 is more resistant to therapy than types 2 and 3. In addition, many current treatments for HCV cause undesirable side effects. Therefore, the latest new antiviral therapy is needed. In particular, new anti-viral therapies with fewer unwanted side effects, more effective for the range of HCV genotypes, or schedules that require less complex administration schedules, i.e., fewer doses per day, need.

The present invention provides compositions and methods of treatment useful for treating viral infections (e. G., HCV). Certain compositions and methods of the present invention produce less undesirable side effects, reduce the potential for viral rebound due to more effective, resistant selection for various HCV genotypes, Less complex.

Accordingly, in one aspect the invention provides compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14 And Compound 16, and a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 16. A method of treating HCV infection in a human, comprising administering to the human two or more compounds selected from SEQ ID NO: 16 and a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 16 < / RTI > and a pharmaceutically acceptable salt thereof, to a human in need thereof.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 16. A method of reducing viral load in a human having HCV, comprising administering to the human two or more compounds selected from SEQ ID NO: 16 and a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 16 < / RTI > and a pharmaceutically acceptable salt thereof in a human, comprising administering to the human an effective amount of a compound of the invention.

 In another aspect, the invention provides a method of treating a patient suffering from a condition selected from the group consisting of Compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, The use of at least two compounds selected from compound 15 and compound 16 and the pharmaceutically acceptable salts thereof.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6, 7, 9, or 10 for the prophylactic or therapeutic treatment of a virus (e. At least two compounds selected from Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, and pharmaceutically acceptable salts thereof.

In yet another aspect, the invention provides the use of a composition of the invention for the prophylactic or therapeutic treatment of a virus (e. G., HCV) infection.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, 13, or 14 for the manufacture of a medicament for the treatment of a virus (e. At least two compounds selected from Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, and pharmaceutically acceptable salts thereof.

In another aspect, the invention provides the use of a composition of the invention for the manufacture of a medicament for the treatment of a virus (e. G., HCV) infection in a human.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 7, compound 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16 and pharmaceutically acceptable salts thereof.

In yet another aspect, the invention provides the use of a composition of the invention for the manufacture of a medicament for ameliorating one or more symptoms of a virus (HCV) infection in a human.

In another aspect, the present invention provides a pharmaceutical composition comprising Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and the pharmaceutically acceptable salts thereof.

In another aspect, the invention provides the use of a composition of the invention for the manufacture of a medicament for reducing viral load in a human.

In another aspect, the invention provides compounds 1, 2, 3, 4, 5, 6 for the manufacture of a medicament for reducing the occurrence of HCV subtypes that are resistant to co- , Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 and pharmaceutically acceptable salts thereof.

In another aspect, the invention provides the use of a composition of the present invention for the manufacture of a medicament for reducing the occurrence of HCV subclasses resistant to co-administered oral antiviral agents in humans.

The compositions and methods of the present invention can provide "synergistic" and "synergistic" effects, i.e. the effect achieved when the active ingredient (including two or more combination compounds) are used together, Lt; / RTI >

The compositions and methods of the present invention are beneficial because they provide treatment for a broad range of HCV genotypes and cause less or less severe side effects than current HCV therapies (e.g., including the administration of interferon). In addition, certain combinations of compounds (e.g., compounds 10 and 5, compounds 10 and 6, and compounds 10, 5 and 6) are significantly more potent than those achieved by current therapies (e.g., HCV therapy) Can provide a higher, sustained virological response (SVR). For example, some combinations of compounds may provide an SVR that is greater than about 70% or greater than about 80%.

Justice

Unless otherwise indicated, the following terms and phrases as used herein are taken to indicate the following meanings. It should not be associated with unclearness or lack of clarity that a particular term or phrase is not clearly defined, and the term is used within its ordinary meaning. Where a trade name is used herein, the applicant intends to independently include the trade name product and the active pharmaceutical ingredient (s) of the trade name product.

As used herein, the term "combination compound" refers to compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15 and compound 16. < / RTI >

Compound 1, as used herein, is as follows:

Compound 1 is 5 - ((6- (2,4-bis (trifluoromethyl) phenyl) pyridazin-3-yl) methyl) -2- (2-fluoro-phenyl) -5 H-imidazo [4 , 5- c ] pyridine or 5H-imidazo [4,5-c] pyridine, 5 - [[6- [2,4- bis (trifluoromethyl) phenyl] pyridazin- May also be referred to as 2- (2-fluorophenyl).

Compound 2, as used herein, is as follows:

Compound 2 (2 R, 6 S, 13a R, 14a S, 16a S) - 2- (8- chloro-2- (2- (isopropylamino) thiazol-4-yl) -7-methoxy-quinoline Yloxy) -6- (cyclopentyloxycarbonylamino) -5,16-dioxoctadecahydrocyclopropa [ e ] pyrrolo [1,2- a ] [1,4] diazacyclo Pentadecin-14a-yl (2,6-difluorobenzyl) phosphinic acid.

Compound 3, as used herein, is as follows:

Compound 4, as used herein, is as follows:

Compound 5, as used herein, is as follows:

Compound 6, as used herein, is as follows:

Compound 7, as used herein, is as follows:

Compound 8, as used herein, is as follows:

Compound 9 (diastereomers at P), as used herein, is as follows:

With respect to Compound 9, reference is made to US 7,964,580 and US 2010/0298257 both of which are incorporated herein by reference for the preparation and purification of Compound 9.

As used herein, Compound 10 (S-isomer of Compound 9) is as follows:

With respect to Compound 10, reference is made to US 7,964,580 and US 2010/0298257 both of which are incorporated herein by reference for the preparation and purification of Compound 10.

As used herein, < RTI ID = 0.0 > 11 < / RTI &

With reference to compound 11, reference is made to US 2010/0081628, which is incorporated herein by reference, for the preparation and purification of compound 11.

Compound 12 (diastereomers at P), as used herein, is as follows:

With reference to compound 12, reference is made to US 20110015146, which is incorporated herein by reference, for the preparation and purification of compound 12. [

As used herein, compound 13 (S-diastereomer of compound 12 at P) is as follows:

With regard to compound 13, reference is made to US 20110015146, which is incorporated herein by reference, for the preparation and purification of compound 13.

Compound 14, as used herein, is as follows:

With reference to compound 14, reference is made to US 7,964,580, which is incorporated herein by reference, for the preparation and purification of compound 14.

Compound 15, as used herein, is as follows:

With reference to Compound 15, reference is made to US 7,964,580, which is incorporated herein by reference, for the preparation and purification of Compound 15. [

Compound 16, as used herein, is as follows:

With reference to compound 16, reference is made to US 7,429,572, which is incorporated herein by reference, for the preparation and purification of compound 16.

With regard to ribavirin, reference is made to EP 0 093 401 B1, which is incorporated herein by reference for its preparation and nomenclature for ribavirin. As used herein, ribavirin refers to:

.

.

Ribavirin is preferably selected from the group consisting of 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, 1- β-D-ribofuranosyl- Carboxyamide; 1 -? - D-ribofuranosyl-1,2,4-triazole-3-carboxamide; Roche; DRG-0028; HSDB 6513; ICN 1229; Megaribavirin (for example, a formulation of 100 mg of ribavirin / mL); NSC 163039; BioPartners; Schering-Plow (Aesca; Bayer Schering Pharma; Essex; Pfizer; Trading Pharma; Zuellig Pharma); Ribamide; Ribamidyl (Biopharma, Russia); Three Rivers Pharmaceuticals; Ribavirin; Ribavirin; Trivinyl; Valeant Pharmaceuticals (ICN Pharmaceuticals); ICA Pharmaceuticals (Alfa Wassermann); Valeant Pharmaceuticals; And Uci-farma (Sao Bernardo do Campo, Sao Paulo, Brazil). Also, as used herein, ribavirin includes analogs of ribavirin, including tarribavirin (bramamidine, ICN 3142).

The term "interferon" is used interchangeably with 1) interferons such as PEGylated rIFN-alpha 2b (PEG-Intron, Merck & Co., Inc., PEGASYS, Hoffmann- La Roche Inc.) (Roferon-A, Hoffmann-La Roche Inc.), interferon alpha (MULTIFERON® Viranative AB Corporation, OPC-18, Alfaferone , Alfanative, subalin, interferon alpha-1 (Valeant), interferon alpha-n1 (WellferonTM, Glaxo Wellcome), interferon alpha-n3 (ALFERON-Hemispherx Biopharma, Inc.), interferon- Biogen Idec, DL-8234 Daiichi Pharmaceutical Co. Ltd.), interferon-omega (omega DUROS®, Alza Corporation, Intarcia Therapeutics, Inc.; Biomed 510, Intarcia Therapeutics, Inc.), ALBUFERON®- Genome Sciences, INC.), IFN alpha-2b XL, BLX-883 (LOCTERON®, Biolex Therapeutics, INC.), DA-3021, glycosylated interferon alpha-2b (AVI-005), PEG-INFERGEN en, Inc., PEGylated interferon lambda- 1 (Type III) (PEGylated IL-29), and BELEROFON®, Nautilus Biotech.

The term " combination therapy "means a composition or method or use comprising two or more combination compounds, and the like. Combination therapies may include, but are not limited to, ribavirin, interferon, alpha-glucosidase 1 inhibitor, hepatoprotectant, and other active ingredients in addition to two or more combination compounds, including: , Toll-like receptor (TLR) -7 agonist, cyclophilin inhibitor, HCV viral entry inhibitor, HCV mature inhibitor, and HCV IRES inhibitor.

The term "active ingredient" encompasses any combination of compounds, ribavirin, interferon, alpha-glucosidase I inhibitor, hepatoprotectant, TLR-7 agonist, cyclophilin inhibitor, HCV viral entry inhibitor, HCV mature inhibitor ≪ / RTI > and an HCV IRES inhibitor, which is capable of exerting or exerting a pharmaceutical effect.

The term " treatment "and its grammatical equivalents, when used in the context of treating a disease, refers to alleviating or halting progression of the disease, or ameliorating the symptoms of one or more diseases, more preferably, And the like. For example, HCV patients may experience improvement in one or both of the following symptoms that may be associated with HCV infection: increased levels of alanine aminotransferase (ALT), heat, headache, muscle pain, jaundice, fatigue , Loss of appetite, nausea, vomiting and diarrhea. Treatment of hepatitis C virus infection can include reducing HCV viral load in HCV-infected humans.

Certain of the compounds described herein may contain one or more chiral centers, or else may exist as multiple stereoisomers. The scope of the present invention includes mixtures of stereoisomers and purified enantiomers or enantiomerically / diastereomerically enriched mixtures. The individual isomers of the compounds represented by the formulas herein and any mixtures thereof fully or partially equilibrated are also included within the scope of the present invention. The present invention also encompasses the individual isomers of the compounds represented by the formulas herein as mixtures with isomers of which one or more chiral centers are inverted. The stereochemical definitions and practices used herein are generally those described in the following references [S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York]; And Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York.

Many organic compounds are present in their optically active form, i. E. They have the ability to rotate the plane of plane polarized light. In the base of an optically active compound, the prefixes D and L or R and S are used to denote the absolute arrangement of molecules with respect to its chiral center (s). The prefixes d and l or (+) and (-) are used to designate the indication of the rotation of the plane polarized light by the compound, and (-) or l means the compound is left-handed. Compounds with the prefix (+) or d are preferential.

Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomer mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate and may occur where there is no stereogenic or stereospecificity during the chemical reaction or process. The terms "racemic mixture" and "racemate" refer to equimolar mixtures of two enantiomeric species that are not optically active.

Combination

The present invention includes combinations of two or more combination compounds. Possible 2 of the compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, Table I, which shows a combination of circles (combination 1-21), 3 circles (combination 22-56), 4 circles (combination 57-92), and 5 circle (combination 93-113), is provided below. Compound 4, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 are nucleoside inhibitors of HCV NS5b polymerase and the combination of combination compounds is Compound 4, Compound 9 , Compound 10, compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16 (see column I, column 6).

[Table 1]

Composition

One aspect of the present invention is a composition comprising Compound 1 and comprising Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, 15 and compound 16, for example, a pharmaceutical composition. In one specific aspect of the invention, the second compound may be Compound 2, Compound 3, Compound 4, Compound 5 or Compound 6.

Another aspect of the present invention is a compound of formula (I) which comprises compound 2 and is selected from compounds 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15 and compound 16, for example, a pharmaceutical composition. In one specific aspect of the present invention, the second compound may be compound 4. [ In one specific aspect of the present invention, the second compound may be compound 3. [ In one specific aspect of the present invention, the second compound may be compound 5.

Another aspect of the present invention is a compound represented by formula (I), which comprises compound 3 and is selected from compounds 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15 and compound 16, for example, a pharmaceutical composition. In one specific aspect of the present invention, the second compound may be Compound 1. [ In one specific aspect of the present invention, the second compound may be compound 4. [ In one specific aspect of the present invention, the second compound may be compound 5. In one specific aspect of the present invention, the second compound may be compound 6.

Yet another aspect of the present invention provides a method for preparing a compound of formula I, comprising adding a first compound selected from the group consisting of compounds 4 and a second compound selected from the group consisting of compounds 1, 2, 3, 5, 6, For example, a pharmaceutical composition. In one specific aspect of the present invention, the second compound may be Compound 1 or Compound 2 or Compound 3 or Compound 6. In one specific aspect of the present invention, the second compound may be Compound 1. [ In one specific aspect of the present invention, the second compound may be compound 2. [ In one specific aspect of the present invention, the second compound may be compound 3. [ In one specific aspect of the present invention, the second compound may be compound 5. In one specific aspect of the present invention, the second compound may be compound 6.

Another aspect of the present invention is a compound 5 comprising Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, 15 and compound 16, for example, a pharmaceutical composition. In one specific aspect of the present invention, the second compound may be Compound 1. [ In one specific aspect of the present invention, the second compound may be compound 6.

Another aspect of the present invention is a composition comprising Compound 6, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, 15 and compound 16, for example, a pharmaceutical composition. In one specific aspect of the present invention, the second compound may be Compound 1. [ In one specific aspect of the present invention, the second compound may be compound 2. [ In one specific aspect of the present invention, the second compound may be compound 3. [ In one specific aspect of the present invention, the second compound may be compound 4. [

Another aspect of the present invention is a composition comprising Compound 7, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, 15 and compound 16, for example, a pharmaceutical composition.

Another aspect of the present invention includes a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, 3, Compound 5, Compound 6 and Compound 7, for example, a pharmaceutical composition.

Another aspect of the present invention is a process for the preparation of a compound of formula I, which comprises compound 1 and is selected from compounds 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, further comprising a second compound and a third compound each selected from the group consisting of compounds 15 and 16, respectively. The second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6. [ The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 2, and the third compound may be compound 3. [ The second compound may be compound 2, and the third compound may be compound 5. [ The second compound may be compound 3, and the third compound may be compound 5. [

Another aspect of the present invention is a compound of formula (I) which comprises compound 2 and is selected from compounds 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, further comprising a second compound and a third compound selected from the group consisting of Compound (I) The second compound may be compound 4. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 5. [ The second compound may be compound 3, and the third compound may be compound 6. [

Another aspect of the present invention is a compound represented by formula (I), which comprises compound 3 and is selected from compounds 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, further comprising a second compound and a third compound selected from the group consisting of Compound (I) The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 4, and the third compound may be compound 5. [

Yet another aspect of the present invention is a process for preparing a second compound comprising a first compound selected from the group consisting of compounds 4 and each selected from the group consisting of compounds 1, 2, 3, 5, 6, A composition further comprising a third compound, for example a pharmaceutical composition. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [ The second compound may be compound 1, and the third compound may be compound 5. [ The second compound may be compound 2, and the third compound may be compound 5. [

Another aspect of the present invention is a compound 5 comprising Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, further comprising a second compound and a third compound selected from the group consisting of Compound (I) The second compound may be compound 1.

Another aspect of the present invention is a composition comprising Compound 6, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, further comprising a second compound and a third compound selected from the group consisting of Compound (I) The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 1, and the third compound may be compound 5. [ The second compound may be compound 2, and the third compound may be compound 5. [ The second compound may be compound 3, and the third compound may be compound 5. [

Another aspect of the present invention is a composition comprising Compound 7, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, further comprising a second compound and a third compound selected from the group consisting of Compound (I)

Another aspect of the present invention includes a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, 3, compound 5, compound 6, and compound 7, respectively, such as a pharmaceutical composition.

Another aspect of the present invention is a process for the preparation of a compound of formula I, which comprises compound 1 and is selected from compounds 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound and a fourth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be Compound 3, Compound 4, Compound 5, or Compound 6. The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 2, the third compound may be compound 4, and the fourth compound may be compound 6. [ The second compound may be compound 3, the third compound may be compound 4, and the fourth compound may be compound 6. [

Another aspect of the present invention is a compound of formula (I) which comprises compound 2 and is selected from compounds 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound and a fourth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be compound 4. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention is a compound represented by formula (I), which comprises compound 3 and is selected from compounds 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound and a fourth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 4, the third compound may be compound 5, and the fourth compound may be compound 6. [

A further aspect of the present invention is a method for preparing a compound of the formula (I), which comprises a first compound selected from the group consisting of compounds 4 and is selected from the group consisting of compounds 1, 2, 3, 5, 6, For example, a pharmaceutical composition, further comprising a third compound and a fourth compound. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [

Another aspect of the present invention is a compound 5 comprising Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound and a fourth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be compound 1.

Another aspect of the present invention is a composition comprising Compound 6, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound and a fourth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 4. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [

Another aspect of the present invention is a composition comprising Compound 7, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound and a fourth compound, respectively, selected from the group consisting of 15 and 16, respectively.

Another aspect of the present invention includes a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, 3, compound 5, compound 6 and compound 7, a third compound, and a fourth compound, respectively, selected from the group consisting of Compound 3, Compound 5, Compound 6 and Compound 7, for example a pharmaceutical composition.

Another aspect of the present invention is a process for the preparation of a compound of formula I, which comprises compound 1 and is selected from compounds 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound, a fourth compound and a fifth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6. The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention is a compound of formula (I) which comprises compound 2 and is selected from compounds 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound, a fourth compound and a fifth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be compound 4. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention is a compound represented by formula (I), which comprises compound 3 and is selected from compounds 1, 2, 4, 5, 6, 7, 9, 10, 11, 12, 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound, a fourth compound and a fifth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

A further aspect of the present invention is a method for preparing a compound of the formula (I), which comprises a first compound selected from the group consisting of compounds 4 and is selected from the group consisting of compounds 1, 2, 3, 5, 6, For example, a pharmaceutical composition, further comprising a third compound, a fourth compound, and a fifth compound. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [

Another aspect of the present invention is a compound 5 comprising Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound, a fourth compound and a fifth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be compound 1.

Another aspect of the present invention is a composition comprising Compound 6, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound, a fourth compound and a fifth compound, respectively, selected from the group consisting of 15 and 16, respectively. The second compound may be Compound 1, Compound 2, Compound 3 and Compound 4. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [

Another aspect of the present invention is a composition comprising Compound 7, wherein Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, For example, a pharmaceutical composition, which further comprises a second compound, a third compound, a fourth compound and a fifth compound, respectively, selected from the group consisting of 15 and 16, respectively.

Another aspect of the present invention includes a first compound selected from the group consisting of Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, For example, a pharmaceutical composition, which further comprises a second compound, a third compound, a fourth compound and a fifth compound each selected from the group consisting of Compound 3, Compound 5, Compound 6 and Compound 7.

salt

Combination compounds and other active ingredients may be present in the form of a salt. Typically, but not absolutely, the salts of the combination compounds and other active ingredients are pharmaceutically acceptable salts. Salts included in the term " pharmaceutically acceptable salts "refer to non-toxic salts of the combination compounds and / or other active ingredients. Suitable examples of pharmaceutically acceptable salts include inorganic acid addition salts such as chloride, bromide, sulfate, phosphate and nitrate; Organic acid addition salts such as acetate, galactarate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate and ascorbate Acid salts; Salts with acidic amino acids, such as aspartate and glutamate; Alkali metal salts such as sodium salts and potassium salts; Alkaline earth metal salts such as magnesium salts and calcium salts; Ammonium salts; Organic basic salts such as trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt and N, N'-dibenzylethylenediamine salt; And salts with basic amino acids such as lysine salts and arginine salts. The salt may in some cases be a hydrate or an ethanol solvate.

Pharmaceutical formulation

Combination compounds and / or other active ingredients may be formulated with conventional carriers or excipients, which may be selected according to conventional practice. Tablets usually contain excipients, fluidizers, fillers, binders, and the like. Aqueous formulations may be prepared in sterile form and will generally be isotonic if delivered by a route other than oral. All formulations will optionally contain excipients, for example, the excipients listed in the Handbook of Pharmaceutical Excipients (1986), the entire contents of which are incorporated herein by reference. Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, and the like.

The pH of the formulation ranges from about 3 to about 11, but is typically about 7 to 10.

While it is possible for one active ingredient to be administered alone, it may be desirable to provide one or more active ingredients as a pharmaceutical formulation. The formulations of the invention (both animal and human) comprise one or more active ingredients together with one or more acceptable carriers and optionally other therapeutic ingredients. The carrier (s) should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and being physiologically harmless to the recipient thereof.

Formulations include those suitable for the routes of administration set forth below. The formulations may conveniently be presented in unit dosage form and may be prepared by any method known in the pharmaceutical arts. Techniques and formulations are generally found in Remington ' s Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.), Whose specialty is incorporated herein by reference. The method comprises bringing into association a combination of an active ingredient with a carrier which constitutes one or more accessory ingredients. In general, the formulations may be prepared by uniformly and directly combining one or more active ingredients with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units, for example capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; Powder or granules; A solution or suspension in an aqueous or non-aqueous liquid; Or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, soft or paste.

Tablets may optionally be made by compression or molding with one or more accessory ingredients. Compressed tablets may be prepared by compressing the active ingredient in a free-flowing form such as powder or granules, optionally in admixture with a binder, lubricant, inert diluent, preservative, surfactant or dispersant, and in a suitable machine. Molded tablets may be prepared by molding in a suitable machine a mixture of powdered active ingredients that is soaked with an inert liquid diluent. Tablets may optionally be coated or scored and optionally formulated to provide sustained release or controlled release of the active ingredient.

For administration to the eye or other external tissues such as mouth and skin, the formulation may contain, for example, 0.075-20% w / w (0.1% w / w increase, e.g. 0.6% w / active ingredient (s) in the range of 0.1% to 20%, preferably 0.2 to 15% w / w, and most preferably 0.5 to 10% w / w, May be preferably applied as a topical ointment or cream containing the component (s). When formulated as ointments, the active ingredient may be used as a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated with a cream having an oil-in-water cream base.

If desired, the aqueous phase of the cream base may contain, for example, a polyhydric alcohol of 30% w / w or more, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and Polyethylene glycol (including PEG 400), and mixtures thereof. Topical formulations may preferably include a compound that enhances absorption or penetration of the active ingredient through the skin or other sites. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

The oil phase of the emulsion of the combination compound and / or other active ingredients can be constituted from known ingredients in a known manner. The phase may comprise only an emulsifier (also known as an emulsifier), but preferably comprises a mixture of both fat or oil, or both fat and oil, and one or more emulsifiers. Preferably, the hydrophilic emulsifier is included with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both oil and fat. The emulsifier (s) together with the stabilizer (s) or without the stabilizer (s) make the so-called emulsified wax and the wax together with the oils and fats make up the so-called emulsified ointment base which forms the oil phase of the cream formulation.

Suitable emulsifiers and emulsion stabilizers for use in the formulation of the present invention include Tween 60 (ICI Americas Inc.), Span 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate .

The choice of a suitable oil or fat for the formulation will depend on achieving the desired cosmetic properties. The cream will preferably be a greasy, colorless and washable product with a suitable density to prevent leakage from the tube or other container. But are not limited to, linear or branched, mono- or dibasic alkyl esters such as di-iso adipate, isocetyl stearate, propylene glycol diesters of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl A combination of branched chain esters known as stearate, 2-ethylhexyl palmitate or Crodamol CAP can be used, with the three at the end being the preferred esters. They may be used alone or in combination depending on the required characteristics. Alternatively, high melting lipids such as white soft paraffin and / or liquid paraffin or other mineral oils may be used.

The pharmaceutical formulations according to the present invention comprise one or more activities with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. The pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended mode of administration. When used for oral use, for example, tablets, troches, cachets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, which may contain one or more pharmaceutically acceptable excipients, including sweetening, flavoring, coloring and preserving agents, May contain the above agonists. Tablets containing the active ingredient are admixed with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. The excipient may be, for example, an inert diluent, for example, calcium carbonate or sodium carbonate, lactose, lactose monohydrate, sodium croscarmellose, povidone, calcium phosphate or sodium phosphate; Granulating and disintegrating agents, for example corn starch, or alginic acid; Binders, for example, cellulose, microcrystalline cellulose, starch, gelatin or acacia; And lubricants, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated by known techniques, including microencapsulation to delay degradation and adsorption in the gastrointestinal tract, thus providing a sustained action over a long period of time. For example, time delay materials such as glyceryl monostearate or glyceryl distearate may be used alone or in combination with the wax.

Formulations for oral use include hard gelatine capsules wherein the active ingredient (s) is mixed with a solid diluent, for example, calcium phosphate or kaolin, or soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil May also be provided as capsules.

The aqueous suspensions of the present invention contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. The excipient may be selected from the group consisting of suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum, and dispersing or wetting agents, Condensates of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensates of ethylene oxide with long chain aliphatic alcohols (for example, heptadecaethyleneoxyethylene (For example, polyoxyethylene sorbitan monooleate), condensates of ethylene oxide with unsaturated esters derived from fatty acids and hexitol anhydrides. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.

The oil suspension may be formulated by suspending the active ingredient in a vegetable oil, for example peanut oil, olive oil, sesame oil or palm oil, or a mineral oil, for example liquid paraffin. Oral suspensions may contain thickening agents, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents such as those set forth herein may be added to provide a tasty oral preparation. The composition may be preserved by the addition of an antioxidant, such as ascorbic acid.

Dispersible powders and granules of the present invention suitable for the manufacture of aqueous suspensions by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, such as sweeteners, flavoring agents and coloring agents, may also be provided.

The pharmaceutical composition of the present invention may be present in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, for example olive oil or peanut oil, a mineral oil, for example liquid paraffin or a mixture thereof. Suitable emulsifying agents include naturally occurring gums such as acacia gum and tragacanth gum, incomplete esters derived from naturally occurring phospholipids, for example soybean lecithin, esters or fatty acids and hexitol anhydrides, such as sorbitan monooleate, And condensates of said incomplete esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated using sweetening agents, for example, glycerol, sorbitol or sucrose. The formulations may also contain emollients, preservatives, flavoring agents or coloring agents.

The pharmaceutical compositions of the present invention may be in the form of sterile injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions. The suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents mentioned herein. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent, or a solution in a solvent such as 1,3-butane-diol or may be prepared as a lyophilized powder . Acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils can be routinely used as a solvent or suspending medium. For this purpose, any of the bland fixed oils may be used including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may also be used in the preparation of injectables.

The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration. For example, a time-release formulation for oral administration to humans comprises from about 1 to about 1000 mg, mixed with a suitable and convenient amount of a carrier material, which may vary from about 5 to about 95% total composition (weight by weight) Of the active substance. The pharmaceutical compositions may be formulated to provide an easily measurable amount for administration. For example, an aqueous solution for intravenous infusion may contain from about 3 to about 500 μg of active ingredient per milliliter (ml) of solution to allow injection of the appropriate volume at a rate of about 30 mL / hr .

Formulations suitable for ocular administration include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in the formulation at a concentration of 0.5 to 20%, advantageously 0.5 to 10%, especially about 1.5% w / w.

Formulations suitable for topical administration in the mouth include lozenges, usually sucrose and acacia or tragacanth, containing the active ingredient of the flavor base; Pastilles comprising the active ingredient of an inert base, such as gelatin and glycerin, or sucrose and acacia; And mouthwashes comprising the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppository with a base comprising a suitable base, for example cocoa butter or salicylate.

Formulations suitable for intrapulmonary or nasal administration may have a particle size, for example in the range of from 0.1 to 500 mu m (inclusive of particle sizes in the range of from 0.1 to 500 mu m, increasing as 0.5 mu m, 1 mu m, 30 mu m, 35 mu m, , By rapid inhalation through the nasal cavity or by inhalation through the mouth to reach the alveoli. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for spray or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as those previously used in the treatment or prevention of infections as described herein.

Formulations suitable for vaginal administration may be presented as pills, tampons, creams, gels, pastes, foams or spray formulations, as well as formulations in which the active ingredient is added to such carriers as are known in the art as appropriate.

 Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacterial growth inhibitors and solutes which provide isotonic formulations with the blood of the intended receptor; And aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be provided in the form of a lyophilized liquid carrier, for example, lyophilized (E.g., freeze-dried under the above-mentioned conditions). Temporary injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage forms may be in the form of a daily dose or unit daily sub-dose of the active ingredient, as herein above recited, or a fraction containing a suitable fraction thereof.

In particular for the addition to the above-mentioned ingredients, the formulations of the combination compounds and / or other active ingredients may comprise other agents customary in the art in connection with the type of formulation in question, for example suitable for oral administration It will be appreciated that the formulations may include flavoring agents.

Combination compounds and other active ingredients may also be formulated to provide controlled release of the active ingredient for less frequent administration or to improve the pharmacokinetics or toxicity profile of the active ingredient. Accordingly, the present invention also provides compositions comprising two or more combination compounds formulated for sustained or controlled release.

Dose

The effective amount of the active ingredient will depend, at least, on the nature of the condition to be treated, the toxicity, whether the compound is used prophylactically (lower dosage) or against the active disease or condition, the delivery method and the pharmaceutical formulation, And may be determined by the clinician using a stepwise escalation study.

By way of example, the compositions of the present invention (e. G., Tablets) may be formulated to provide an effective amount. For example, with respect to Compound 1, or a pharmaceutically acceptable salt thereof, the composition may comprise 1.0 mg to 100 mg, 5 mg to 40 mg, 30 mg to 50 mg, or 20 mg or 40 mg Any one or more of Compound 2, Compound 3, Compound 6, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, Can be adjusted to be administered to a human being at least once a day. With respect to Compound 2, or a pharmaceutically acceptable salt thereof, the composition may comprise 25 mg to 800 mg, 50 mg to 400 mg, or 60 mg to 300 mg or 70 mg to 200 mg, Can be combined with any one or more of Compound 1, Compound 3, Compound 6, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, To be administered to a human in need thereof at least once a day. With respect to Compound 3, or a pharmaceutically acceptable salt thereof, the composition may comprise from 10 mg to 1000 mg, or from 50 mg to 400 mg, or from 100 mg to 400 mg, or from 200 mg to 400 mg, It is also possible to use the above compound 1, 2, 6, 4, 5, 7, 9, 10, 11, 12, 13, 14, May be adjusted to be administered at least once a day. With respect to Compound 4, or a pharmaceutically acceptable salt thereof, the composition may comprise from 25 mg to 400 mg or from 25 mg to 200 mg, and any one or more of Compound 1, Compound 2, Compound 3, Compound 6, Compound 5 and Compound 7 can be combined to be administered to a human in need thereof at least once a day. With respect to Compound 5, or a pharmaceutically acceptable salt thereof, the composition may comprise 50 mg to 1000 mg or 100 mg to 750 mg, and any one or more of Compound 1, Compound 2, Compound 3, Compound 6, Compound 4, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 can be adjusted to be administered to a human in need thereof more than once a day. With respect to Compound 6, or a pharmaceutically acceptable salt thereof, the composition may comprise 1 mg to 500 mg or 3 mg to 300 mg or 3 mg to 200 mg or 3 mg to 100 mg or 10 mg to 90 mg, And at least one of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 And Compound 16 can be adjusted to be administered to a human in need thereof at least once a day. With respect to Compound 7, or a pharmaceutically acceptable salt thereof, the composition may comprise 100 μg to 3000 mg or less, 25 mg to 2000 mg or less, or 50 mg to 1000 mg or less, and any one or more compounds 1 , Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, (For example, four times a day). With respect to compounds 9 and 10, or a pharmaceutically acceptable salt thereof, the composition may comprise from 10 mg to 1000 mg per day (according to US 2010/0298257). With respect to Compound 11, or a pharmaceutically acceptable salt thereof, the composition may comprise from 1 mg to 1000 mg per day (according to US 2010/0081628). Doses for co-administered compounds 1-7 may need to be adjusted to account for potential drug-drug interactions. For example, although Compound 1 did not appear to affect the drug metabolism system, Compound 2 appears to have an effect of increasing the exposure of Compound 1 by about 2-3X. Thus, when Compound 1 is used in combination with Compound 2, it is possible to expect a reduction in the dose of Compound 1 (for example, 2x-3x). When combined with compound 16, compound 2 appears to have an effect of increasing the exposure of compound 6 by approximately 5x, and thus, when compound 16 is administered with compound 2, a dose reduction of compound 16 (e. G., 3x-5x ) Can be expected. Thus, when co-administered with Compound 2, the 10 mg dose of Compound 6 is similar to the 30 mg dose.

Two or more combination compounds may be administered with ribavirin in an amount of about 800 mg, 1000 mg or 1200 mg per day, with single or multiple doses (e.g., twice daily, about 400 mg, 500 mg or 600 mg) .

Use of the combination of the present invention

In practicing these aspects of the invention, the combination compound can be used at the dosages indicated above.

One aspect of the present invention includes Compound 1 for use in a method of treating HCV infection wherein Compound 1 is Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16. The second compound is selected from the group consisting of compound 11, compound 12, compound 13, The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.

Another aspect of the present invention includes compound 2 for use in a method of treating HCV infection, wherein compound 2 is Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16. The second compound is selected from the group consisting of compound 11, compound 12, compound 13, The second compound may be compound 4.

Another aspect of the present invention includes compound 3 for use in a method of treating HCV infection wherein compound 3 is Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16. The second compound is selected from the group consisting of compound 11, compound 12, compound 13, The second compound may be Compound 1 or Compound 6.

Another aspect of the present invention includes compound 4 for use in a method of treating HCV infection, wherein compound 4 is selected from the group consisting of compounds 1, 2, 3, 5, 6, Is used in combination with the compound. The second compound may be Compound 1 or Compound 2 or Compound 3 or Compound 6.

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infection wherein Compound 5 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16. The second compound is selected from the group consisting of compound 11, compound 12, compound 13, The second compound may be compound 1.

Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infection wherein Compound 6 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16. The second compound is selected from the group consisting of compound 11, compound 12, compound 13, The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.

Another aspect of the present invention includes compound 7 for use in a method of treating HCV infection wherein Compound 7 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16. The second compound is selected from the group consisting of compound 11, compound 12, compound 13,

Another aspect of the present invention includes a compound 9 for use in a method of treating HCV infection wherein compound 9 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes a compound 10 for use in a method of treating HCV infection, wherein the compound 10 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes a compound 11 for use in a method of treating HCV infection, wherein the compound 11 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes a compound 12 for use in a method of treating HCV infection, wherein the compound 12 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes a compound 13 for use in a method of treating HCV infection, wherein the compound 13 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes a compound 14 for use in a method of treating HCV infection wherein compound 14 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes compound 15 for use in a method of treating HCV infection, wherein compound 15 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes a compound 16 for use in a method of treating HCV infection, wherein the compound 16 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, 2 < / RTI > compound.

Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infection wherein Compound 1 is Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16, respectively. The second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6. [ The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention includes compound 2 for use in a method of treating HCV infection, wherein compound 2 is Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16, respectively. The second compound may be compound 4. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention includes compound 3 for use in a method of treating HCV infection wherein compound 3 is Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16, respectively. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the invention includes compound 4 for use in a method of treating HCV infection, wherein compound 4 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound and the third compound. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infection wherein Compound 5 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16, respectively. The second compound may be compound 1.

Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infection wherein Compound 6 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16, respectively. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [

Another aspect of the present invention includes compound 7 for use in a method of treating HCV infection wherein Compound 7 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16, respectively.

Another aspect of the invention includes a compound 9 for use in a method of treating HCV infection wherein compound 9 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound and the third compound.

Another aspect of the present invention includes a compound 10 for use in a method of treating HCV infection wherein compound 10 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound and the third compound.

Another aspect of the present invention includes a compound 11 for use in a method of treating HCV infection wherein compound 11 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound and the third compound.

Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infection wherein Compound 1 is Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16 are used in combination with the second compound, the third compound and the fourth compound, respectively. The second compound may be Compound 3, Compound 4, Compound 5, or Compound 6. The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention includes compound 2 for use in a method of treating HCV infection, wherein compound 2 is Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, compound 12, compound 13, compound 14, compound 15 and compound 16 are used in combination with the second compound, the third compound and the fourth compound, respectively. The second compound may be compound 4. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention includes compound 3 for use in a method of treating HCV infection wherein compound 3 is Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound The second compound, the third compound, and the fourth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the invention includes compound 4 for use in a method of treating HCV infection, wherein compound 4 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound, the third compound and the fourth compound. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infection wherein Compound 5 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound The second compound, the third compound, and the fourth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be compound 1.

Another aspect of the present invention includes Compound 6 for use in a method of treating HCV infection wherein Compound 6 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Compound The second compound, the third compound, and the fourth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be compound 4, and the third compound may be compound 6. [ The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [

Another aspect of the present invention includes compound 7 for use in a method of treating HCV infection wherein Compound 7 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Compound The second compound, the third compound, and the fourth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16.

Another aspect of the invention includes a compound 9 for use in a method of treating HCV infection wherein compound 9 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound, the third compound and the fourth compound.

Another aspect of the present invention includes a compound 10 for use in a method of treating HCV infection wherein compound 10 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound, the third compound and the fourth compound.

Another aspect of the present invention includes a compound 11 for use in a method of treating HCV infection wherein compound 11 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound, the third compound and the fourth compound.

Another aspect of the present invention includes Compound 1 for use in a method of treating HCV infection wherein Compound 1 is Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound The second compound, the third compound, the fourth compound, and the fifth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6. The second compound may be compound 2, and the third compound may be compound 4. [ The second compound may be compound 3, and the third compound may be compound 4. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention includes compound 2 for use in a method of treating HCV infection, wherein compound 2 is Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound The second compound, the third compound, the fourth compound, and the fifth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be compound 4. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the present invention includes compound 3 for use in a method of treating HCV infection wherein compound 3 is Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound The second compound, the third compound, the fourth compound, and the fifth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 4. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 4, and the third compound may be compound 6. [

Another aspect of the invention includes compound 4 for use in a method of treating HCV infection, wherein compound 4 is selected from the group consisting of compounds 1, 2, 3, 5, 6, 2 compound, the third compound, the fourth compound and the fifth compound. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1, and the third compound may be compound 3. [ The second compound may be Compound 1, and the third compound may be Compound 6. [ The second compound may be compound 2, and the third compound may be compound 6. [ The second compound may be compound 3, and the third compound may be compound 6. [

Another aspect of the present invention includes Compound 5 for use in a method of treating HCV infection wherein Compound 5 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9, Compound 10, Compound The second compound, the third compound, the fourth compound, and the fifth compound selected from the group consisting of Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16. The second compound may be compound 1.

Another aspect of the invention includes compound 6 for use in a method of treatment of an HCV infection wherein Compound 6 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9, Compound 10, Is used in combination with the second compound, the third compound, the fourth compound and the fifth compound each selected from the group consisting of the compound 11, the compound 12, the compound 13, the compound 14, the compound 15 and the compound 16. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4. The second compound may be compound 1 and the third compound may be compound 2. [ The second compound may be compound 1 and the third compound may be compound 3. [ The second compound may be compound 4 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 4. [ The second compound may be compound 3 and the third compound may be compound 4. [

Another aspect of the invention includes compound 7 for use in a method of treatment of HCV infection wherein compound 7 is Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9, Compound 10, Is used in combination with the second compound, the third compound, the fourth compound and the fifth compound each selected from the group consisting of the compound 11, the compound 12, the compound 13, the compound 14, the compound 15 and the compound 16.

Another aspect of the invention includes a compound 9 for use in a method of treatment of HCV infection wherein compound 9 is selected from the group consisting of compound 1, compound 2, compound 3, compound 5, compound 6 and compound 7 The second compound, the third compound, the fourth compound and the fifth compound.

Another aspect of the present invention includes compound 10 for use in a method of treatment of HCV infection wherein compound 10 is selected from the group consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7 The second compound, the third compound, the fourth compound and the fifth compound.

Another aspect of the invention includes compound 11 for use in a method of treatment of an HCV infection wherein compound 11 is selected from the group consisting of compound 1, compound 2, compound 3, compound 5, compound 6 and compound 7 The second compound, the third compound, the fourth compound and the fifth compound.

One aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 1 and further comprising administering Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound < RTI ID = 0.0 > 9, compound 10, compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16. The second compound is selected from the group consisting of: The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 2 and further comprising administering Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, and Compound 16. [0064] The second compound may be compound 4.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 3 and further comprising administering Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, and Compound 16. [0064] The second compound may be Compound 1 or Compound 6.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 4 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 ≪ / RTI >

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 5 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, and Compound 16. [0064] The second compound may be compound 1.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 6 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, and Compound 16. [0064] The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 7 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, and Compound 16. [0064]

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 9 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 ≪ / RTI >

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 10 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 ≪ / RTI >

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 11 and further comprising administering to the patient a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7 ≪ / RTI >

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 1 and further comprising administering Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, respectively. The second compound may be Compound 3, or Compound 4, or Compound 5 or Compound 6. [ The second compound may be compound 2 and the third compound may be compound 4. [ The second compound may be compound 3 and the third compound may be compound 4. [ The second compound may be compound 2 and the third compound may be compound 6. [ The second compound may be compound 3 and the third compound may be compound 6. The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 2 and further comprising administering Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, respectively. The second compound may be compound 4. The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 3 and further administering Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10 , Compound 11, compound 12, compound 13, compound 14, compound 15, and compound 16, respectively. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. [ The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 4 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 Lt; RTI ID = 0.0 > and / or < / RTI > The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be compound 1 and the third compound may be compound 2. [ The second compound may be compound 1 and the third compound may be compound 3. [ The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 6. [ The second compound may be compound 3 and the third compound may be compound 6.

 Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 5 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, respectively. The second compound may be compound 1.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 6 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, respectively. The second compound may be Compound 1, Compound 2, Compound 3 or Compound 4. The second compound may be compound 1 and the third compound may be compound 2. [ The second compound may be compound 1 and the third compound may be compound 3. [ The second compound may be compound 4 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 4. [ The second compound may be compound 3 and the third compound may be compound 4. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 7 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9 , Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16, respectively.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 9 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 Lt; RTI ID = 0.0 > and / or < / RTI >

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 10 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 Lt; RTI ID = 0.0 > and / or < / RTI >

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 11 and further comprising administering to the patient a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7 Lt; RTI ID = 0.0 > and / or < / RTI >

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 1 and further comprising administering Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be Compound 3, Compound 4, Compound 5, or Compound 6. The second compound may be compound 2 and the third compound may be compound 4. [ The second compound may be compound 3 and the third compound may be compound 4. [ The second compound may be compound 2 and the third compound may be compound 6. [ The second compound may be compound 3 and the third compound may be compound 6. The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 2 and further comprising administering Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. [ The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 3 and further comprising administering Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. [ The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 4 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 The second compound, the third compound, and the fourth compound, respectively. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 6. The second compound may be compound 1 and the third compound may be compound 2. [ The second compound may be compound 1 and the third compound may be compound 2. [ The second compound may be compound 1 and the third compound may be compound 3. [ The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 6. [ The second compound may be compound 3 and the third compound may be compound 6.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 5 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be compound 1.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 6 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be Compound 1, Compound 2, Compound 3, or Compound 4. The second compound may be Compound 1, and the third compound may be Compound 2. [ The second compound may be compound 1 and the third compound may be compound 3. [ The second compound may be compound 4 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 4. [ The second compound may be compound 3 and the third compound may be compound 4. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 7 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 9 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 The second compound, the third compound, and the fourth compound, respectively.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 10 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 The second compound, the third compound, and the fourth compound, respectively.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 11 and further comprising administering to the patient a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7 The second compound, the third compound, and the fourth compound, respectively.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 1 and further comprising administering Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be Compound 3, Compound 4, Compound 5 or Compound 6. The second compound may be compound 2 and the third compound may be compound 4. [ The second compound may be compound 3 and the third compound may be compound 4. [ The second compound may be compound 2 and the third compound may be compound 6. [ The second compound may be compound 3 and the third compound may be compound 6. The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 2 and further comprising administering Compound 1, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be compound 4. The second compound may be Compound 1 and the third compound may be Compound 4. [ The second compound may be compound 1 and the third compound may be compound 6. The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 3 and further comprising administering Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be Compound 1 or Compound 6. The second compound may be Compound 1 and the third compound may be Compound 4. [ The second compound may be Compound 1 and the third compound may be Compound 4. [ The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 4 and the third compound may be compound 6. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 4 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 The third compound, the fourth compound, and the fifth compound, respectively, which are selected from the group consisting of: The second compound may be Compound 1, Compound 2, Compound 3 or Compound 6. The second compound may be compound 1 and the third compound may be compound 2. [ The second compound may be compound 1 and the third compound may be compound 3. [ The second compound may be compound 1 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 6. [ The second compound may be compound 3 and the third compound may be compound 6.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 5 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 6, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be compound 1.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 6 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 7, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively. The second compound may be Compound 1, Compound 2, Compound 3, and Compound 4. The second compound may be compound 1 and the third compound may be compound 2. [ The second compound may be compound 1 and the third compound may be compound 3. [ The second compound may be compound 4 and the third compound may be compound 6. [ The second compound may be compound 2 and the third compound may be compound 4. [ The second compound may be compound 3 and the third compound may be compound 4. [

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 7 and further comprising administering Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 9 , The compound 10, the compound 11, the compound 12, the compound 13, the compound 14, the compound 15, and the compound 16, respectively.

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 9 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 The third compound, the fourth compound, and the fifth compound, respectively, which are selected from the group consisting of:

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 10 and further comprising administering a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6, and Compound 7 The third compound, the fourth compound, and the fifth compound, respectively, which are selected from the group consisting of:

Another aspect of the invention is a method of improving one or more symptoms of an HCV infection in a human, a method of reducing viral load in a human diagnosed with HCV, a method of treating HCV in a human subject, Wherein each method comprises administering Compound 11 and further comprising administering to the patient a therapeutically effective amount of a compound consisting of Compound 1, Compound 2, Compound 3, Compound 5, Compound 6 and Compound 7 The third compound, the fourth compound, and the fifth compound, respectively, which are selected from the group consisting of:

Route and route of administration

Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 11, Compound 12, Compound 13, Compound 14, Compound 15 and Compound 16 May be adapted to be administered by any route appropriate to the condition being treated. Suitable routes include, but are not limited to, oral, rectal, nasal, topical (including nasal and sublingual), vaginal and parenteral (subcutaneous, intramuscular, intravenous, intradermal, intracerebral and epidural). It will be appreciated that the preferred path may be varied, for example, depending on the condition of the recipient.

A synergistic effect can be obtained when the active ingredient is: (1) co-formulated (e. G. In a single dosage form) and administered or delivered simultaneously in a combined formulation; (2) delivered alternately or in parallel as separate formulations; Or (3) by some other curing method. When delivered in an alternation therapy, a synergistic effect can be obtained when the compound is administered or delivered in order, for example, as a separate tablet, pill or capsule or by different infusion into a separate syringe. Generally, during an alternation therapy, the effective dose of each active ingredient is administered sequentially, i. E., Sequentially, while in combination therapy an effective dosage of two or more active ingredients is administered together.

Co-administration of a combination compound and one or more combination compounds generally refers to simultaneous or sequential administration of one or more combination compounds, such that a therapeutically effective amount of two or more combination compounds is present in the patient. In some cases, a combination compound (e. G., 2, 3 or 4 combination compounds) will be co-formulated to allow simultaneous administration. In some cases, co-formulated combination compounds may be co-formulated with one or more additional combination compounds.

Co-administration also includes administration of a unit dose of the combination compound before or after administration of a unit dose of one or more other active ingredients, for example, a combination of two or more within a few seconds, minutes or hours of administration of one or more other active ingredients ≪ / RTI > For example, a unit dose of the combination compound is administered first, followed by administration of a unit dose of the second combination compound within a few seconds or minutes, and administration of a unit dose of one or more other active ingredients within a few seconds or minutes You can follow. Alternatively, unit doses of one or more other active ingredients may be administered first, followed by administration of a unit dose of the combination compound within a few seconds or minutes, and administration of a unit dose of the second combination compound within a few seconds or minutes Can be followed. In some cases, a unit dose of the combination compound is first administered, followed by administration of a unit dose of the second combination compound after a period of several hours (e.g., 1-12 hours), after a period of several hours Lt; / RTI > for 1 to 12 hours), followed by administration of a unit dose of one or more other active ingredients. In other cases, a unit dose of one or more other active ingredients is first administered, followed by administration of a unit dose of the combination compound after a period of several hours (e.g., 1-12 hours), after a period of several hours Lt; RTI ID = 0.0 > 1-12 < / RTI > hours), followed by administration of a unit dose of the second combination compound. When more than two combination compounds are administered with one or more additional active ingredients, the combination compounds may be administered sequentially in a few seconds, minutes or hours (e.g., 1-12 hours) with each other, and one or more additional active ingredients May be administered before, during or after administration of the combination compound. When the combination compound is co-formulated, it may be administered concurrently with, or before or after administration of one or more additional active ingredients.

Unless otherwise indicated, combination therapies may be administered in separate dosage forms separate from each other, administered together or separately, sequentially or simultaneously, and at a time close to or close to each other.

The course of treatment may extend, for example, from about 12 weeks to about 48 weeks, or longer, for example from about 12 weeks to about 24 weeks.

The present invention includes combinations of therapeutically active ingredients to ameliorate one or more symptoms of HCV infection in humans including, without limitation, nausea, vomiting, anorexia, fatigue, jaundice, vomiting, diarrhea, dehydration, abdominal pain and cirrhosis. In addition, the use of combination therapy in some HCV infected individuals is effective in reducing the viral load of HCV viral particles present in the infected person's body to a statistically significant amount. For example, viral load can be measured, for example, by measuring plasma HCV RNA levels using a COBAS TaqMan HCV assay (Roche Molecular Systems). Typically, an HCV infected human being treated with a combination compound according to the present invention experiences an improvement in one or both of the symptoms associated with HCV infection.

Combinations of two or more combinations of ribavirin but not interferon

As discussed above, some current HCV therapies include administration of interferon, but such treatment typically produces undesired side effects. Thus, it would be desirable to find an effective HCV treatment that does not require the administration of interferon.

One aspect of the present invention provides compositions, methods, uses, etc., for the treatment of HCV, comprising administering two or more combination compounds, or a pharmaceutically acceptable salt thereof, and ribaviryl, without administration of one or more interferon . This aspect of the present invention may be particularly useful because it allows effective treatment of HCV without the side effects associated with the administration of one or more interferons.

In one aspect of the invention, the combination amount of ribavirin and a combination compound or a pharmaceutically acceptable salt thereof, optionally one or more additional agents, is effective in treating an HCV infection.

Another aspect of the invention includes a method of ameliorating one or more symptoms of a human HCV infection, comprising: administering to the patient a combination of a compound or a pharmaceutically acceptable salt thereof and at least two of the < RTI ID = 0.0 > ≪ / RTI > In this regard, the present invention does not exclude the potential for administering one or more interferons. Moreover, the present invention may indeed be used in combination with another therapy comprising one or more interferons. Aspects of the invention include the effective treatment of HCV by ribavirin that does not require one or more interferons.

Another aspect of the invention includes a method of reducing viral load in a human diagnosed with HCV, comprising: administering two or more combination compounds or a pharmaceutically acceptable salt thereof and ribavirin without one or more interferon .

Another aspect of the invention includes a method for treating HCV in a human subject comprising administering ribavirin essentially in combination with at least two of the combination compounds or a pharmaceutically acceptable salt thereof.

Another aspect of the invention includes a method of reducing the occurrence of HCV subclasses resistant to coadministered oral antiviral agents, comprising: administering two or more combination compounds, or a combination thereof, Administering an acceptable salt and ribavirin.

Similarly, another aspect of the invention is a composition for improving one or more symptoms of HCV infection in a human comprising at least two combination compounds or a pharmaceutically acceptable salt thereof and ribavirin without one or more interferon, Gt; composition. ≪ / RTI > Another aspect of the present invention includes a composition for reducing viral load in a human diagnosed with HCV comprising at least one combination compound or a pharmaceutically acceptable salt thereof and ribavirin without one or more interferon. Another aspect of the invention includes a composition for treating HCV in a human subject comprising ribavirin essentially in combination with two or more combination compounds or a pharmaceutically acceptable salt thereof. Another aspect of the present invention includes a composition for ribavirin-based HCV therapy comprising two or more combination compounds or a pharmaceutically acceptable salt thereof, provided that the composition does not comprise one or more interferon. Yet another aspect of the present invention provides a composition for reducing the occurrence of HCV subclasses resistant to coadministered oral antiviral agents, comprising at least one combination compound or a pharmaceutically acceptable salt thereof and ribavirin without one or more interferon .

Similarly, another aspect of the invention includes: in the manufacture of a medicament for alleviating one or more symptoms of an HCV infection in a human, in the absence of one or more interferon, a combination of two or more combination compounds or a pharmaceutically acceptable salt thereof and a ribavirin The use of; And the use of two or more combination compounds or a pharmaceutically acceptable salt thereof and ribavirin in the manufacture of a medicament for reducing viral load in a human diagnosed with HCV, without one or more interferon; And the use of ribavirin in combination with two or more combination compounds or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of HCV in a human subject, said use not involving the use of one or more interferons; And the use of two or more combination compounds or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for ribavirin-based HCV therapy, wherein said administration avoids administration of one or more interferons; And the use of two or more combination compounds or a pharmaceutically acceptable salt thereof and ribavirin without one or more interferon in the manufacture of a medicament for reducing the occurrence of HCV strains resistant to co-administered oral antiviral agents.

Another aspect of the present invention includes a combination comprising ribavirin and two or more combination compounds or a pharmaceutically acceptable salt thereof, wherein the combination is substantially free of one or more interferon. In one aspect, the combinations may occur in separate dosage forms separate from each other, administered together or separately, sequentially or simultaneously, and at near or at a remote time to each other.

Another aspect of the present invention includes a kit comprising: a ribavirin, a combination of two or more compounds and instructions for treatment curing methods for treatment, viral load reduction, or delayed onset or progression of HCV, The administration of two or more combination compounds and ribavirin without the administration of one or more interferons). In one embodiment, the kit may also include a package, e.g., a blister pack. Alternatively, the kit may provide separate dosages of the respective components and separate prescriptions separately from the packaged drug, but may be combined with instructions for treatment curing methods for treatment, reduction of viral load or delayed onset or progression of HCV The above is intended to be within the scope of the present invention.

Another aspect of the invention includes a pharmaceutical composition comprising: ribavirin; At least two combination compounds, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may be in a single dosage form.

Unless otherwise specified, combination therapies with ribavirin may be administered in separate dosage forms from each active ingredient (including combination compounds) administered, and may be administered together (e. G., In unit dosage form, such as tablets) , Sequentially or simultaneously, and close to each other or at a remote time. When administered separately, each compound may be administered concurrently with the other (s), or with the other (s) before or after. The active ingredient may be administered daily. In one embodiment, the daily dose of the active ingredient is administered daily at separate sub-doses, such as once, twice or three times, or four times. Advantageously, the daily dose of the combination compound or its pharmaceutically acceptable salt and ribavirin can be administered once daily.

Although the present invention provides a pharmaceutical composition comprising two or more combination compounds or a pharmaceutically acceptable salt thereof without interferon; And ribavirin, the present invention does not exclude the potential for administering one or more interferons to a human. Moreover, the invention may in fact be used in conjunction with another therapy for another guideline comprising one or more interferons.

A combination of two or more combination compounds and ribavirin and interferon

Another aspect of the present invention provides compositions, methods, uses, and the like comprising administering two or more combination compounds or a pharmaceutically acceptable salt thereof and ribavirin and one or more interferon for the treatment of HCV. Administration of more interferon may be a temporal relationship to administration of the combination compound and ribavirin.

Another aspect of the invention includes a method of ameliorating one or more symptoms of an HCV infection in a human, comprising administering two or more combination compounds or a pharmaceutically acceptable salt thereof, ribavirin, and one or more interferon. Another aspect of the invention includes a method of reducing viral load in a human diagnosed with HCV comprising: administration of ribavirin and one or more interferons in combination with two or more combination compounds or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention includes a method of ribavirin-based HCV therapy comprising administration of ribavirin and one or more interferon together with two or more combination compounds or a pharmaceutically acceptable salt thereof.

Another aspect of the invention includes a method of reducing the occurrence of HCV subclasses resistant to coadministered oral antiviral agents comprising: administering to a patient in need of such treatment a therapeutically effective amount of ribavirin and < RTI ID = 0.0 > Administration of one or more interferons.

Another aspect of the invention includes the use of two or more combination compounds or a pharmaceutically acceptable salt thereof, and one or more interferons in the manufacture of a medicament for ameliorating one or more symptoms of an HCV infection in a human. Another aspect of the present invention involves the use of ribavirin and one or more interferons in combination with two or more combination compounds or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing viral load in a human diagnosed with HCV. Another aspect of the present invention includes the use of ribavirin in combination with two or more combination compounds or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of HCV in a human subject wherein said use comprises the use of one or more interferons . Another aspect of the present invention includes the use of two or more combination compounds, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for ribavirin-based HCV therapy, wherein said administration comprises administration of one or more interferon. Another aspect of the present invention relates to a pharmaceutical composition comprising a combination of two or more combination compounds or a pharmaceutically acceptable salt thereof, ribavirin, and one or more compounds of the invention in the manufacture of a medicament for reducing the occurrence of HCV subclasses resistant to co- Interferon < / RTI >

Another aspect of the present invention includes a combination comprising ribavirin and two or more combination compounds, or a pharmaceutically acceptable salt thereof, wherein the combination comprises one or more interferons.

Another aspect of the present invention includes a kit comprising: ribavirin, two or more combination compounds and one or more interferons; And instructions for treatment regimens for treatment, reduction of viral load or delaying the onset or progression of HCV, wherein said therapeutic regimen comprises administration of two or more combination compounds and administration of ribavirin and administration of one or more interferon. In one aspect, the kit may also include a package, e.g., a blister pack. Alternatively, the kit may provide a dose of each component as an individual prescription and separately packaged drug, but may be combined with instructions for treatment regimens for treatment, reduction of viral load, or delay of initiation or progression of HCV The above is considered to be within the scope of the present invention.

Another aspect of the present invention includes a pharmaceutical composition comprising: at least two combination compounds or a pharmaceutically acceptable salt thereof, ribavirin, and one or more interferons; And one or more pharmaceutically acceptable carriers. In one aspect, the pharmaceutical composition may be in a single dosage form.

Unless otherwise specified, combination therapies with ribavirin and one or more interferons may be administered in separate dosage forms from one or more interferons administered to the patient, and each of the remaining active ingredients desired to be used in combination therapy (including combination compounds) Are administered together (e. G., In unit dosage form, e. G. Tablets) or separately, sequentially or simultaneously and close to each other or at a remote time. When administered separately, each active ingredient may be administered concurrently with the administration of the other (s), or with the other (s) before or after the administration. The active ingredient may be administered daily. In one embodiment, the daily dose is administered in a separate sub-dose, e. G. Once, twice, three or four times daily.

Combination therapy, including adjunctive therapy

In another embodiment, a non-limiting example of a suitable combination includes at least two compounds 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, , Compound 14, compound 15 and compound 16 and one or more additional active ingredients HCV NS3 protease inhibitor, alpha-glucosidase I inhibitor, hepato protectant, nucleoside or nucleotide inhibitor of HCV NS5B polymerase, HCV NS5A inhibitor, HCV NS5B inhibitor, TLR-7 agonist, cyclophilin inhibitor, HCV IRES inhibitor, HCV entry inhibitor, HCV mature inhibitor, HCV assembly inhibitor, HCV infection inhibiting factors and pharmacokinetic enhancers, and other drugs for the treatment of HCV. More specifically, one or more compounds of the present invention may be combined with one or more compounds selected from the group consisting of:

(i) HCV NS3 protease inhibitors such as Boseferprile (SCH-503034, SCH-7), Telaprevir (VX-950), TMC-435 (IUPAC N - [(2R, 3aR, Methoxy-8-methylquinolin-4-yloxy] -5-methyl-4, 14-di < / RTI > Oxo-1,2,3,3a, 4,5,6,7,8,9,11a, 12,12a, 13,14,14a-hexadecahydrocyclopenta [c] cyclopropa [g] [1 , 6] diazacyclotetradecyne-12a-carbonyl] cyclopropanesulfonamide], ABT-450, ACH-1625, ACH-2684, BI- 201335, BI-1230, MK- YH-5531, YH-5531, and ITMN-191 (R-910), VY-3700, 7227);

(ii) alpha-glucosidase inhibitors such as selcognivir (MX-3253), UT-231B, miglitol;

(iii) hepatoprotectants such as emericasan (IDN-6556), ME-3738, sialylin and MitoQ;

(iv) nucleoside or nucleotide inhibitors of HCV NS5B polymerase such as R1626, R7128 (R4048), IDX184, IDX-102, PSI-661, PSI-938, PSI- BCX-4678, valproicitabine (NM-283), MK-0608 and TMC649128;

(v) a non-nucleoside inhibitor of HCV NS5B polymerase, such as, for example, filibbil (PF-868554), ABT-333, ABT-072, BI-207127, VCH- A-48747, BC-2329, VCH-796 (Nessbubir), JTK-652, MK-3281, VBY-708, VCH-222, A848837, ANA-598, GL60667, GL59728, A- , GSK625433, BILN-1941, and XTL-2125;

(vi) HCV NS5A inhibitors such as ACH-2928, AZD-2836 (A-831), AZD-7295 (A-689), BMS-766, BMS-790052, BMS-824393 and PPI- ;

(vii) TLR-7 agonists such as, for example, imiquimod, 852A, ANA-773, ANA-975, AZD-8848 (DSP-3025), PF-04878691 and SM-360320 and compound 8;

(viii) cyclophilin inhibitory factors, such as DEBIO-025, SCY-635, and NIM811;

(ix) HCV IRES inhibitory factors, such as MCI-067;

(x) a pharmacokinetic enhancer such as, for example, loxithromycin, BAS-100, SPI-452, PF-4194477, TMC-41629;

(xi) HCV entry inhibiting factor

(xii) an inhibitor of HCV assembly;

(xiii) HCV maturation inhibitory factor;

(xiv) inhibitors of HCV infection; And

(xv) Other drugs for the treatment of HCV, such as thymosin alpha 1, Alinea NTZ, BIVN 401 virostat, PYN-17 altirex, KPE02003002, actilon (CPG-10101), KRN-7000, civacir, GI-5005, XTL-6865, BIT225, PTX-111, ITX2865, TT-033i, ANA 971, NOV-205, taraxin, EHC FGF-188, VGX-410C, EMZ-702, AVI 4065, BMS-650032, BMS-791325, Bavituximab, MDX-1106 (ONO-4538), Oglufanide, , And VX-497 (merimeepodib).

Synthetic example

Synthetic protocols for the preparation of compounds 1, 2, 3, 6, 7 and 8 are known in the literature. Additionally, synthetic protocols for the preparation of each of the combination compounds are provided in the Examples below.

Compound 1 can be prepared using synthetic methods and intermediates such as those described in US 7,754,720. Compound 1 can also be prepared as described in the following examples.

Example 1 : 5 - ({6- [2,4-Bis (trifluoromethyl) phenyl] pyridazin-3-yl} methyl) -2- (2- fluorophenyl) , 5-c] pyridine 1 < / RTI >

Compound 103 was dissolved in dimethoxyethane (DME). To this solution was added 2,4-bis (trifluoromethyl) phenylboronic acid 105 and an aqueous 2N Na ₂ CO ₃ solution was added. Pd (PPh ₃₎ To the resulting two-phase mixture after the addition of ₄ and the reaction was heated at 80 ℃ for 72 hours. The reaction was cooled to room temperature, filtered through celite, and the celite was washed with EtOAc. The filtrate was concentrated in vacuo. The residue was purified on 6 g of SiO ₂ using MeOH / CH ₂ Cl ₂ to elute the compound. The compound thus obtained was contaminated with PPh ₃ (O). The product was refilled in a 1 mM Chromatotron plate using 0- > 5% MeOH / CH ₂ Cl ₂ in 1% steps. The pure fractions were combined, concentrated in vacuo, and then dried under high vacuum for 12 hours. 11.8 mg of the free base of Compound 1 was obtained without PPh ₃ contamination.

Intermediate compound 104 was prepared as follows.

a. Preparation of Compound ( 102 )

To a solution of commercially available starting material 101 in CHCl ₃ was added trichloroisocyanuric acid (TCCA) at 60 < 0 > C. The solution was then stirred for 1.5 hours, cooled, and filtered through HiFlo-Celite. The filtrate was concentrated and dried under vacuum. 5.037 g of Compound 102 was obtained.

b. Preparation of Compound 104

To a solution of 103 in DMF (dimethylformamide) was added NaOH. Compound 102 was dissolved in DMF (20 ml) and slowly added to the solution. The reaction was stirred for 3 h, diluted with water and extracted with EtOAc. The organic layer was dried over Na ₂ SO ₄ . The solvent was removed and the product was recrystallized from dichloromethane. 5.7 g of compound 103 was obtained.

Compound 2 can be prepared using synthetic methods and intermediates such as those described in USSN 12/202319 (US 20100051763 A1). Compound 2 can also be prepared as described in the following examples.

Example 2 : Preparation of compound 2

The phosphinate ester 206 (23.7 g, 24.05 mmol) was dissolved in CH ₃ CN (240 mL) and cooled to 0 占 폚. Iodotrimethylsilane (17.4 mL, 122.3 mmol) was added at a rapid dropping rate and after 10 minutes 2,6-lutidine (17.0 mL, 146.4 mmol) was added dropwise. The reaction mixture was slowly warmed to room temperature, stirred for 1 hour, then cooled to 0 < 0 > C and 2,6-lutidine (11.1 mL, 95.6 mmol) followed by MeOH (24 mL). The solution was concentrated in vacuo and the crude residue was purified by HPLC to give 12.68 g of compound 2 in 55% yield.

Intermediate Compound 206 was prepared as follows.

a. Preparation of Compound 203

Compound 201 (17.42 g, 28.30 mmol) was dissolved in THF (136 mL) and cooled to 0 < 0 > C. To the solution was added N-methylmorpholine (4.7 mL, 42.7 mmol). After 10 min at 0 C, i-butyl chloroformate (4.05 mL, 30.96 mmol) was added dropwise. After an additional hour, a solution of (1-amino-2-vinyl- cyclopropyl) - (2,6-difluoro-benzyl) -phosphinic acid ethyl ester 202 (8.94 g, 29.70 mmol) in THF Was slowly added as a solution. The suspension was allowed to warm to room temperature and after 2 h it was partitioned between H ₂ O (400 mL) and ethyl acetate (200 mL). The aqueous layer was extracted with ethyl acetate (200 mL x 2) and the combined organic layers were extracted with HCl (IN, 225 mL) and H2O (200 mL). The acid wash and aqueous washings were combined and extracted again with ethyl acetate (175 mL x 2, 100 mL x 2). The combined organic layers were washed with brine (400 mL), dried over Na2SO4 and concentrated in vacuo to yield 25.06 g of diene 203 in 98.5% crude yield. LCMS (M + 1): 898.06.

b. Preparation of compound 204 .

Compound 203 (12.91 g, 14.36 mmol) was dissolved in CH ₂ Cl ₂ (1440 mL) and the solution was degassed for 30 minutes. The solution was heated to 40 < 0 > C and Grubb's G1 catalyst (2.95 g, 3.59 mmol) was added. Tris-hydroxymethylphosphine (22.3 g, 18.0 mmol), TEA (50 mL, 35.9 mmol) and H ₂ O (400 mL) were added and the reaction mixture was stirred for additional 16 h Lt; / RTI > The reaction mixture was cooled to room temperature and the two layers were separated. The organic layer was washed with H ₂ O (400 mL) and brine (300 mL), dried over MgSO 4 and concentrated. The crude residue was purified by silica-gel chromatography to give 8.30 g of macrocyclic olefin 204 in 66% yield. LCMS (M + 1): 870.09.

c. Preparation of Compound 205

The macrocyclic olefin 204 (7.34 g, 8.42 mmol) was dissolved in ethyl acetate (105 ml) and rhodium / aluminum (5 wt%, 2.945 g, 0.40 wt%) was added. The system was emptied and flushed to H ₂ (1 atm, 3x). The system was charged with more rhodium / aluminum (5 wt%, 842 mg, 0.10 wt%) after 3 hours, emptied and flushed with H ₂ (1 atm, 3x). After an additional 1 hour, the suspension was filtered and concentrated in vacuo to yield 6.49 g of reduced Macrocycle 205 in 88% crude yield. LCMS (M + 1): 872.04.

d. Preparation of compound 206 .

N- methylpyrrolidinone (25.0 mL) to the blower chamber mark rate was dissolved in cycle 205 (6.49 g, 7.67 mmol) , 8- Chloro-2- (2-isopropylamino-thiazol-4-yl) -7 -methoxy-quinolin-4-ol 207 (2.564 g, 7.33 mmol) after the _{Cs 2 CO 3 (4.40 g,} 13.50 mmol) was added. The mixture was heated to 65 [deg.] C for 6 hours, then diluted with ethyl acetate (200 mL) and washed with LiCl (5%, 250 mL). The aqueous layer was extracted with ethyl acetate (100 mL x 2) and the combined organic layers were washed with brine (150 mL), dried over Na ₂ SO ₄ / MgSO ₄ and concentrated in vacuo. The crude residue was purified via silica-gel chromatography (ethyl acetate-methanol) to give 4.39 g of aminothiazole 206 in 58% yield. LCMS (M + 1): 985.28.

Intermediate compound 201 can be prepared as follows.

e. Preparation of compound 209

Compound 208 (7.00 g, 28.55 mmol) and DABCO (5.13 g, 45.94 mmol) were dissolved in toluene (30 mL). A solution of brothyl chloride (10.22 g, 40.01 mmol) in toluene (11 mL) was added. The reaction mixture was stirred overnight at room temperature. The reaction was diluted with EtOAc (210 mL) and 0.5N HCl (200 mL) was added. The two layers were separated and the aqueous layer was extracted with EtOAc (2 x 200 mL). The combined organic layers were washed with brine (200 mL), dried over Na ₂ SO ₄ , filtered and concentrated. The crude product was purified by combi-flash, yielding 12.23 g of compound 209 in 92% yield.

f. Preparation of compounds 210 and 212

Compound 209 (12.2 g, 26.3 mmol) was treated with 4 N HCl / 1,4-dioxane (60 mL) and stirred for 1 hour. The reaction mixture was concentrated and dried under vacuum for 20 minutes. The crude amine HCl salt of compound 210 was dissolved in DMF (150 mL) and acid 211 (14.2 g, 52.6 mmol) was added. HATU (20.0 g, 52.6 mmol) and NMM (13.5 g, 131.5 mmol) were added. The reaction mixture was stirred overnight at room temperature. The reaction was diluted with EtOAc (300 mL), washed with 1 N HCl (200 mL), saturated NaHCO ₃ , brine, dried over Na ₂ SO ₄ and concentrated. The crude product was purified by comb-flash to yield 15.1 g of compound 212 in 93% yield.

g. Preparation of Compound 213

To a solution of 212 (12.8 g, 20.7 mmol) in CH ₂ Cl ₂ (50 mL) was added 4N HCl in 1,4-dioxane (50 mL, 200 mmol). The reaction mixture was stirred at room temperature for 2 hours, concentrated and dissolved in dried under vacuum for 20 _{min, CH 3 CN (50 mL)} . Saturated NaHCO ₃ in H ₂ O (50 mL) was added and stirred for 5 min. Cyclopentyl chloroformate in freshly prepared THF (50 mL) was added. The reaction was completed within one hour. The solvent was removed under reduced pressure and the residue was diluted with EtOAc. The mixture was made pH = 2 with 1 N HCl and the two layers were separated. The organic layer was washed with brine, dried over Na ₂ SO _4, filtered and concentrated to yield the crude compound 213 (3.18 g).

h. Preparation of Compound ( 201 ).

The crude ester 213 (3.18 g, 5.07 mmol) was dissolved in THF (25 mL), H ₂ O (25 mL) and then MeOH (6 mL) and LiOH (660 mg, 25.4 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour and diluted with EtOAc. The reaction mixture was acidified to pH 2 using 1 N HCl and the two layers were separated. The aqueous layer was extracted with EtOAc (2 x). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , concentrated and dried under vacuum to yield 3.09 g of the acid 201 .

Intermediate 8-Chloro-2- (2-isopropylamino-thiazol-4-yl) -7-methoxy-quinolin-4-ol 207 can be prepared as follows.

i. Preparation of 8-chloro-4-hydroxy-7-methoxyquinoline-2-carboxylic acid 215

MeOH: 1 of THF: To a solution of methyl 8-chloro-4-hydroxy-7-methoxyquinoline-2-carboxylate 214 (36.5g, 0.145 mol) of the first mixture (total 160 mL), H ₂ 0 A solution of LiOH (30.5 g, 0.725 mol) in tetrahydrofuran (80 mL) was added. When the LCMS analysis showed complete conversion to the carboxylic acid, the mixture was stirred at room temperature for one hour. The reaction was terminated by removing volatiles and adjusting the pH of the solution to 6 with aqueous 6N HCl. The resulting tasted residue was filtered and dried in a freeze dryer for 2 days to yield 34.4 g (99.6%) of 215 as a white solid. EI MS (m / z) 253.9 [M + H].

j. Preparation of 2- (2-diazo-1-oxo) -8-chloro-7-methoxyquinolin-4-ylisobutylcarbonate 216 .

To a solution of 8-chloro-4-hydroxy-7-methoxyquinoline-2-carboxylic acid 215 (10.2 g, 0.04 mol) in THF (400 mL) was added triethylamine (12.3 mL , 0.088 mol) and i -butyl chloroformate (11.6 mL, 0.088 mol). When the LCMS analysis proved the completion of the reaction to provide the desired mixed anhydride, the mixture was stirred for 1 hour at 0 < 0 > C. EI MS (m / z) 454.0 [M + H]. To the reaction mixture of the anhydrous was added a 1M solution of diazomethane (121 mL, 0.121 mol) in diethyl ether at 0 ° C through a plastic funnel. The mixture was allowed to warm to room temperature for an additional 2 hours while stirring. Analysis of the mixture by LCMS proved the completion of the reaction. The diaphragm was removed and the reaction was stirred for an additional 20 minutes before the solvent was removed. The residue was further dried under high vacuum to yield 216 , which was sent to the next step. EI MS (m / z) 377.9 [M + H].

k. 8-chloro-2- (2- (isopropylamino) thiazol-4-yl) -7-methoxyquinoline-4-ol Preparation of 207.

Cooling of 2- (2-diazo-l-oxo) -8-chloro-7-methoxyquinolin-4-ylisobutyl carbonate 216 (15.2 g, 0.040 mol) in THF (268 mL) To the solution was slowly added 48% HBr (23 mL, 0.201 mol) over 15 minutes. If the LCMS analysis demonstrates complete reaction, the solution is stirred at 0 < 0 > C for an additional 40 min. The reaction was terminated by the addition of aqueous 1N NaOH (180 mL) at 0 ° C to adjust the pH of the aqueous layer to 9. The layers were separated and the aqueous layer was washed with EtOAc (2 x 200 mL). The combined organic extracts were washed with brine and dried over MgSO4. The solvent was removed in vacuo to yield 17.7 g of a yellow solid. EI MS (m / z) 4 31 .9 [M + H].

A solution of the bromoketone obtained from the above reaction was suspended in i-propanol (270 mL) and isopropyl isourea (9.4 g, 0.080 mol). The reaction mixture was heated at 72 [deg.] C for 32 hours. LCMS analysis of the reaction proved complete conversion to the desired product. The reaction was cooled to room temperature, allowing the product to precipitate out of solution. The reaction was further cooled to < RTI ID = 0.0 > 0 C < / RTI > The filtrate was washed with ether and dried in a freeze dryer to give 8.03 g of compound 207 as an orange solid.

Compound 3 can be prepared using synthetic methods and intermediates such as those described in USSN 12 / 215,605 (US 20090257978 A1). Compound 3 can also be prepared as described in the following examples.

Example 3: Preparation of Compound 3

Compound 315 (12 g, 13 mmol) was dissolved in THF (200 ml) and LiOH (11 g, 260 mmol) in H ₂ O (200 ml) was added followed by MeOH (200 ml). The mixture was stirred at room temperature for 20 hours. At the completion of the reaction, the pH was adjusted to 7 by the addition of 4N HCl in H ₂ O at 0 ° C. The mixture was extracted with EtOAc (2 x 400 ml). The combined organic layers were washed with brine, dried (Na ₂ SO ₄ ) and concentrated in vacuo to give compound 3 as a yellow solid (11 g, 93%). LC / MS = 911.52 (M ^< + & gt ^; +1).

Intermediate Compound 315 was prepared as follows.

a. Preparation of Compound 301

For drying, anhydrous dichloromethane (100 mL) and Et ₂ Zn (28 mL, 273 mmol) were added to an argon purged 3-neck round bottom flask (1000 mL) at 0 ° C. (Note: the source of argon can not come from the needle, use only a suitable glass adapter, a second bubbler can also be attached to the flask to prevent excessive pressure build up). Then, cyclopenten-3-ol (10.0 mL, 119 mmol) was added dropwise (large amount of ethane gas was produced) to the flask, and the reaction mixture was stirred until generation of gas stopped. Diiodomethane (22 mL, 242 mmol) was added dropwise over a period of 30 minutes thereafter. The reaction was allowed to warm to room temperature and stirring continued overnight under a positive flow of argon, at which point TLC analysis showed complete disappearance of the starting alcohol. The reaction was then diluted with CH ₂ Cl ₂ and quenched with 2 M HCl (the white precipitate had to be completely dissolved). The biphasic mixture was poured into a separatory funnel and the organic layer was collected. The solvent was removed under reduced pressure until 100 mL of the material containing compound 301 remained.

b. Preparation of Compound 302

To the flask was added anhydrous dichloromethane (525 mL) and then triethylamine (34 mL, 245 mmol) was added dropwise. The reaction was continued to stir at room temperature under a clear stream of nitrogen, at which point disuccinimidyl carbonate (40.7 g, 159 mmol) was added to the flask in portions. The reaction was continued to stir (2-3 days) until the TLC analysis showed complete disappearance of the starting material. Upon completion, the reaction mixture was quenched with 1 M HCl (200 mL x 2) and washed with H ₂ O (200 mL x 2). The desired material was extracted with CH ₂ Cl ₂ and the combined organic layers were dried using anhydrous MgSO ₄ and passed through a silica plug. The solvent was removed under reduced pressure and the crude was purified by flash chromatography (R _f = 0.33, 1: 1 Hex / EtOAc) to give compound 302 (22 g, 75%).

c. Preparation of compound 304

N- t -Boc- cis-4-hydroxy -L- proline, methyl ester 303 (100.0 g, 407.7 mmol) and DABCO (1.5eq, 68.6g at 2 L 3- necked round bottom flask equipped with a mechanical stirrer and additional funnel , 611.6 mmol) was dissolved in anhydrous toluene (200 mL). After cooling the solution to 0 ° C under N ₂ , a solution of 4-bromo-benzenesulfonyl chloride (1.3 eq, 135.6 g, 530.0 mmol) in 300 mL of toluene was added via addition funnel over 60 minutes. The reaction mixture was stirred and allowed to warm to room temperature overnight (16 h). The mixture was slowly poured into ₂ L 1M Na ₂ CO ₃ (aq) and the product was extracted with EtOAc (2 L). The organic phase was washed with 0.5 N HCl (2 L), H ₂ O (1 L), and brine (1 L), which was dried (MgSO ₄ ) and concentrated to yield 195.45 g of yellow oily broccylate product.

4.0 M HCl in dioxane (500 mL, 5 eq) was slowly added to a solution of the brothylate (407.7 mmol) in dichloromethane (300 mL) and the resulting solution was stirred at room temperature for 2 h. Ether (500 mL) was added to the reaction mixture, then the mixture was stirred for 15 minutes, and the white precipitate was collected by filtration. The solid was washed with ether and hexane and then dried under vacuum overnight to give 153.0 g (381.8 mmol) of the HCl amine salt of compound 304 as a 94% yield (in the case of step 2).

d. Preparation of Compound 305

HATU (217.76 g, 572.7 mmol) and Hunig's base (126 mL, 1145.4 mmol) were added to a solution of Boc- tert -butyl-glycine (97.0 g, 420.0 mmol) in DMF (200 mL) and methylene chloride Respectively. The mixture was stirred at room temperature for 20 minutes and then a solution of HCl salt (153.0 g, 381.8 mmol) and Hunig's base (126 mL, 1145.4 mmol) in DMF (200 mL) and dichloromethane Lt; / RTI > The reaction mixture was stirred at room temperature with LCMS monitoring for 3 h. The reaction mixture was concentrated, dichloromethane was removed under reduced pressure, and the resulting white solid was filtered. The remaining DMF solution was diluted with ethyl acetate (1 L) and washed successively with 3% LiCl (aq) (3x650 mL), saturated NH ₄ Cl (2 x 500 mL), 0.5 N HCl (aq) (2x600 mL), brine (500 mL) ₃ ( ₃ x 500 mL), and brine (500 mL). The resulting organic fraction was dried (MgSO ₄ ) and concentrated to give compound 305 (111 g).

e. Preparation of Compound 306

To a solution of methyl ester 305 (120 g, 207.8 mmol) in THF (300 mL), MeOH (75 mL) was added a solution of LiOH (26.18 g, 623.4 mmol) in H ₂ O (150 mL). The solution was stirred for 4 hours at room temperature. The mixture was acidified to pH about 5.5 with 3N HCl while cooling in an ice-bath, stirred for 10 minutes, and the resulting white solid was collected by filtration. The solid was washed with more water, ether and hexane. The solid was dried under vacuum at 40 < 0 > C overnight to yield 95.78 g (82%) of acid 306 .

f. Preparation of Compound 307

HATU (82.3 g, 216.4 mmol) and Hunig's base (47.5 mL, 432.8 mmol) were added at room temperature to a solution of carboxylic acid 306 (81.4 g, 144.27 mmol) in DMF (200 mL) and dichloromethane (200 mL). After stirring the mixture at room temperature for 20 minutes, a solution of amine (158.7 mmol) and Hunig's base (47.5 mL, 1145.4 mmol) in DMF (200 mL) and dichloromethane (200 mL) was added in one portion to the acid mixture. The reaction mixture was stirred at room temperature for 3 hours and monitored by LCMS. The mixture was concentrated under reduced pressure to remove dichloromethane, and the resulting white solid was filtered. The remaining DMF solution was diluted with ethyl acetate (600 mL) and successively washed with 3% LiCl (aq) (2 x 550 mL), saturated NH ₄ Cl (500 mL), 1N HCl (aq) (500 mL), saturated NaHCO ₃ And brine (300 mL). The resulting organic fraction was dried (Na ₂ SO ₄ ) and concentrated to give compound 307 (111 g).

g. Preparation of compound 308 .

Compound 307 was dissolved in 4N HCl in dioxane (300 mL) at room temperature and stirred for 2 hours. This was then concentrated in vacuo and co-evaporated with dichloromethane (2 x 200 mL) and dried. The residue was dissolved in EtOAc (600mL) and saturated aqueous NaHCO ₃ (1L). This was stirred vigorously. After 10 minutes, the carboxylic acid bicyclo [3.1.0] hex-3-yester ester 2,5-dioxo-pyrrolidin-1-yl ester 302 (41.4 g, 173.1 mmol) was added in one portion. The resulting mixture was stirred for another 30 minutes, collect the organic layer, washed with brine (500 mL), dried (Na ₂ SO _4), and concentrated. The crude product was purified by flash chromatography on silica gel using ethyl acetate / hexanes to give 94.44 g (92%) of compound 308 .

h. Preparation of Compound 310

(2-amino-3-chloro-4-hydroxy-phenyl) -ethanone 309 (70.7 g, 354 mmol) in 48% aqueous HBr (500 mL) was stirred at 110 <0> C for 72 hours. After cooling the mixture to 0 [deg.] C with stirring, the solid was filtered and washed with water. Grinding the resulting solid with a saturated NaHCO ₃ solution (~ 350 mL), filtered, washed with water, and dried under vacuum to give the crude of 310 ~ 40 g (61%) as a dark brown solid. LC / MS = 186 (M ^&lt; + & gt ^; +1).

i. Preparation of Compound 311

(2-amino-3-chloro-4-hydroxy-phenyl) -ethanone 310 (40 g, 215 mmol) was dissolved in DMF (360 ml). Cesium carbonate (140 g, 430 mmol) was added followed by bromoacetaldehyde dimethylacetal (54.5 g, 323 mmol). The mixture was then vigorously stirred at 65 &lt; 0 &gt; C for 24 hours. Upon cooling to room temperature, EtOAc (1 L) and H ₂ O (1 L) were added to the mixture. The organic layer was extracted with EtOAc (1 x 400 ml). The combined organic layers were washed with aqueous 3% LiCl solution (2 x 1 L), brine, dried (Na ₂ SO ₄ ) and concentrated in vacuo. The residue was purified by silica gel chromatography to give 311 as a white solid (39 g, 67%).

j. Preparation of Compound 312

-Ethanone 311 (13 g, 47.5 mmol) and isopropylaminothiazole (2-amino-3-chloro-4- -4-carboxylic acid hydrobromide (12.64 g, 47.5 mmol) was added slowly at -40 &lt; 0 &gt; C phosphorus oxychloride (9.47 g, 61.8 mmol). The mixture was then stirred at &lt; RTI ID = 0.0 &gt; 0 C &lt; / RTI &gt; When the reaction was complete, H ₂ O (30 ml) was added dropwise to the mixture. The mixture was then stirred at 0 &lt; 0 &gt; C for another 15 min. The mixture was concentrated in vacuo. The residue was diluted with EtOAc and was washed with a saturated NaHCO ₃ aqueous solution. Dry the organic layer (Na ₂ SO ₄₎ and concentrated under vacuum. The residue was dissolved in CH ₂ Cl ₂ , hexane was added slowly to the solution, and a yellow solid started to fall off. More hexane was added until the product remained in the mother liquor, yielding compound 312 (18 g, 85%).

k. Preparation of Compound 313

2-Chloro-3- (2,2-dimethoxy-ethoxy) -phenyl] -amide 312 (18 g, 40.7 mmol) was added to a solution of 2-isopropylamino-thiazole- And suspended in toluene (400 ml). H ₂ evolution was monitored while NaH (2.4 g, 61 mmol) was added to the strongly stirred mixture. The mixture became clear during heating for reflux. The reaction was complete after refluxing for 3 hours. The mixture was cooled to room temperature. A solution of AcOH (69.2 mmol) in H ₂ O (3 vol) was added to the mixture. From 0 ℃ After strong agitation for 1 hour, the solids were collected by filtration and was rinsed with H ₂ O. The wet cake was dried under high vacuum to a constant weight to provide compound 313 (15 g, 86%).

l. Preparation of compound 314 .

Cesium carbonate (25.1 g, 77 mmol) was added to a mixture of the brothylate intermediate 303 (15 g, 35 mmol) and compound 313 (27.5 g, 38.5 mmol) in NMP (200 ml). The mixture was stirred at 65 &lt; 0 &gt; C for 5 h. The reaction was cooled to room temperature and an aqueous solution of EtOAc (600 ml) and 3% LiCl (600 ml) was added to the mixture. The organic layer was an aqueous 3% LiCl (1 x 600 ml ), washed with brine, dried (Na ₂ SO _4), and concentrated in vacuo. The residue was purified by silica gel chromatography to give the desired methyl ester as a yellow solid (23.6 g, 75%). LC / MS = 900.13 (M ^&lt; + & gt ^; +1).

m. Preparation of Compound 315

Methyl ester 314 (23.6 g, 26 mmol) was dissolved in glacial acetic acid (200 mL) and 1.4 N HCl in H ₂ O (75 mL) was added to the solution. The mixture was stirred at 60 &lt; 0 &gt; C for 1 hour. When the reaction was complete, the mixture was concentrated to remove the solvent and coevaporate with toluene (x 2) to remove residual acetic acid. The residue was then monitored for CO ₂ release while dissolving in EtOAc (500 ml) and saturated aqueous NaHCO ₃ solution (sufficient to neutralize the mixture). The organic layer was washed with brine, dried (Na ₂ SO _4), concentrated in vacuo. The residue was further dried under high vacuum for 1 hour and used as such in the next step. The crude was dissolved in CH ₂ Cl ₂ (360 ml) and morpholine (3.4 g, 39 mmol) and sodium triacetoxyborohydride (7.2 g, 34 mmol) were added to the mixture at 0 ° C. Glacial acetic acid (0.47 g, 7.8 mmol) was added dropwise to the mixture. The reaction was completed within 10 minutes at 0 ° C. The reaction by the addition of saturated NaHCO ₃ solution was quenched. Further, after stirring for another 20 minutes, the organic layer was washed with brine, dried (Na ₂ SO _4), concentrated in vacuo. The residue was purified by silica gel chromatography to give the desired amine product 315 as a yellow solid (12 g, 50%). LC / MS = 924.63 (M ^&lt; + & gt ^; +1).

Compound 4 can be prepared as described in the following examples.

Example 4 : Preparation of Compound ( 4 ).

The diastereomeric mixture 414 was dissolved in heptane and isopropanol (70%: 30%, 230 mg in 4.5 mL of mixed solvent) and applied to chiral column separation under the following conditions:

Column: Chiralcel OD-H, 2 x 25 cm

Solvent system: 70% heptane and 30% isopropanol

Flow rate: 6 mL / min.

Loading volume per run: 2.5 mL

Compound 4 had a residence time of 20 minutes.

Compound 4 was then recrystallized from MTBE for x-ray quality determination.

Compound 4a had a residence time of 50 minutes.

Intermediate diastereomeric mixture 414 was prepared as follows.

a. Preparation of compound 402 .

To a solution of compound 401 (22.0 g, 54.9 mmol, prepared according to the procedure described in JOC , 2004, 6257) in methanol (300 mL) over a period of 30 min was added dropwise a solution of acetyl chloride ) Was added dropwise, followed by stirring at room temperature for 16 hours. The mixture was concentrated, redissolved in ethyl acetate (400 mL), washed with ice-cold 2N NaOH, concentrated to dryness, and crude methyl ether 402 was obtained as an oil. MS = 437.2 (M + Na &lt; ⁺ &gt;).

b. Preparation of compound 403 .

To a solution of 402 in methanol (300 mL) was added a 0.5 M sodium methoxide solution in methanol (20 mL, 10 mmol) and stirred at room temperature for 16 hours. The reaction was quenched with 4.0 N HCl solution in dioxane (2.5 mL, 10 mmol). The mixture was then concentrated to give crude compound 403 . MS = 201.0 (M + Na &lt; ⁺ &gt;).

c. Preparation of compound 404 .

A mixture of compound 403 , Tritron X-405 (70% in water, 6.0 g), 50% KOH (in water, 85 g) in toluene (500 mL) using an attached Dean-Stark trap Was heated to reflux. After 1 hour of collecting 25 ml of water, benzyl chloride (33 g, 260 mmol) was added and refluxing was continued with stirring for 16 hours. The mixture was then cooled and partitioned between ethyl acetate (400 mL) and water (300 mL). The organic layer was washed with water (300 mL) and concentrated. The residue was purified by silica gel column chromatography (20% EtOAc / hexanes) to give methyl ether 404 as an oil (22.0 g, 89% in step 3).

^{1 H NMR (300 MHz, CDCl} 3): δ7.3 (m, 15H), 4.5-4.9 (m, 7H), 4.37 (m, 1H), 3.87 (d, 1H), 3.56 (m, 2H), 3.52 (s, 3H), 1.40 (s, 3H).

d. Preparation of compound 405 .

To a solution of 404 (22.0 g, 49.0 mmol) in acetic acid (110 mL) was added 3 M sulfuric acid (prepared by mixing 4.8 g of concentrated sulfuric acid and 24 mL of water) and stirred at 70 DEG C for 8 hours . The mixture was concentrated to a volume of 20 mL and partitioned between ethyl acetate and ice-cold 2N NaOH. The ethyl acetate layer was concentrated and purified by silica gel column chromatography (~ 35% EtOAc / hexanes) to give compound 405 as an oil (17.0 g, 80%). MS = 457.2 (M + Na &lt; ⁺ &gt;).

e. Preparation of compound 406 .

To a solution of compound 405 (45 g, 104 mmol) in DMSO (135 mL) was added acetic anhydride (90 mL, 815 mmol) at room temperature under argon. The mixture was stirred at room temperature for 16 hours and then poured into ice-water (1 L) with stirring. After the ice was completely dissolved (30 min), ethyl acetate (500 mL) was added. The organic layer was separated. The extraction procedure was repeated 3 times (3x500 mL). The organic extracts were combined and concentrated. The residue was purified by silica gel column chromatography (20% EtOAc / hexanes) to give 406 as an oil (39 g, 88%).

f. Preparation of compound 407

Pyrrolo [2,1-f] [1,2,4] triazin-4-ylamine (234 mg, 1.10 mmol) (WO2007056170 ) And anhydrous THF (1.5 mL). TMSCl (276 L, 2.2 mmol) was then added and the reaction mixture was stirred for 2 hours. The flask was placed in a dry ice / acetone bath (-78 ° C) and BuLi (2.5 mL, 4.0 mmol, 1.6 M in hexane) was added dropwise. After 1 hour, a solution of compound 406 (432.5 mg, 1.0 mmol) in THF was cooled to 0 &lt; 0 &gt; C and then added dropwise to the reaction flask. After stirring at -78 ℃ 1 hour, it allowed to warm to the flask at 0 ℃ quench the reaction by the addition of saturated NH ₄ Cl (5 mL). The organic layer was extracted with EtOAc (3 x 10 mL) and the combined organic layers were dried over MgSO ₄ . The solvent was removed under reduced pressure and the crude was purified using flash chromatography (hexane / EtOAc). 560 mg (90%) of compound 407 was isolated as a mixture of two anomers.

g. Preparation of compound 408

To a solution of 407 (1 g, 1.77 mmol) in CH ₂ Cl ₂ (20 mL) at 0 ° C was added TMSCN (1.4 mL, 10.5 mmol) and BF ₃ -Et ₂ O (1 mL, 8.1 mmol) . The reaction mixture was stirred at 0 &lt; 0 &gt; C for 0.5 h, then for an additional 0.5 h at room temperature. The reaction was quenched with NaHCO ₃ at 0 ° C and diluted with CH ₃ CO ₂ Et. The organic phase was separated, washed with brine, dried over Na ₂ SO _4, filtered and concentrated. The residue was chromatographed on silica gel and eluted with CH ₃ CO ₂ Et-hexane (1: 1 to 2: 1) to provide 408 (620 mg, 61%) as an isomeric mixture. MS = 576.1 (M + H ^& lt ^{; +} & gt ^; ).

h. Preparation of Compound 409

To a solution of 408 (150 mg, 0.26 mmol) in CH ₂ Cl ₂ (4 mL) at -78 ° C was added BCl ₃ (2 mL, IM in CH ₂ Cl ₂ ). The reaction mixture was stirred at -78 &lt; 0 &gt; C for 1 hour. The reaction was quenched at -78 [deg.] C by dropwise addition of TEA (2 mL) and MeOH (5 mL). The mixture was allowed to warm to room temperature, evaporated and co-evaporated several times with MeOH. The residue was treated with NaHCO ₃ (1 g in 10 mL H ₂ O), concentrated and purified by HPLC to give the desired product 409 (48 mg, 60%).

Other alpha-anomers were also obtained (9 mg, 11%):

i. Preparation of compound 412

Compound 410 (commercially available, 4.99 g, 23.8 mmol) was dissolved in dichloromethane (100 mL) and alanine isopropyl ester hydrogen chloride 411 (3.98 g, 23.8 mmol) was added. The resulting clear solution was cooled at -78 [deg.] C for 30 minutes. Triethylamine (6.63 mL, 47.5 mmol) was added dropwise over 15 minutes. The mixture was allowed to warm to room temperature. After 16 h, the solvent was removed by an argon stream. The residue was redissolved in MTBE (25 mL) and the insoluble material was removed by filtration under argon. The filtrate was condensed with an argon stream and the crude product 412 was used in the next reaction without further purification steps.

j. Preparation of Compound 413

To a solution of 409 (1.03 g, 3.37 mmol) in THF (20 mL) and trimethylphosphate (2.0 mL) was added N -methyl imidazole (1.5 g, 18.3 mmol) at 0 ° C. A solution of 412 (2.5 g, 8.18 mmol) in THF (3 mL) was added dropwise. The resulting mixture was allowed to warm to room temperature over 1.5 h. Partitioned between ethyl acetate and water. The ethyl acetate layer was concentrated and the residue was purified by silica gel chromatography (ethyl acetate-10% ethanol / ethyl acetate) to give 1.15 g (59%) of 413 as a 1: 1 diastereomeric mixture in phosphorus .

k. Preparation of compound 414 .

To a solution of compound 413 (175 mg, 0.305 mmol) in acetonitrile (2 mL) was added N, N -dimethylformamide dimethylacetal (41 μL, 0.34 mmol, 1.1 eq.) And stirred at room temperature for 1 hour . The reaction was complete (LCMS). The mixture was then dried and concentrated. DCC (250 mg, 1.21 mmol, 4 eq.), Acetonitrile (5 mL) and isobutyric acid (55 mg, 58,, 2 eq.) Were added to the residue. The mixture was stirred at room temperature for 48 hours. Water (0.2 mL) and trifluoroacetic acid (0.1 mL) were added at 0 &lt; 0 &gt; C and stirred at room temperature for 64 hours. Sodium bicarbonate (500 mg) was added at 0 &lt; 0 &gt; C. The mixture was stirred at room temperature for 0.5 hour and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (5% methanol / dichloromethane) to give 144 mg (73%) of 414 as a 1: 1 diastereomeric mixture in phosphorus.

Compound 5 was prepared as described in the following examples.

Example 5: Preparation of Compound 5: 5- (3,3-dimethyl-1-boot-yl) -3 - [(cis-4-hydroxy -4 - {[(3 S) - tetrahydro- furan-3-yloxy] methyl} cyclohexyl) {[(1 R) -4- methylcyclohexyl hex-3-en-1-yl] carbonyl} amino] thiophene-2-carboxylic acid 5.

(3R-dimethyl-but-1-ynyl) -3 - [((1R) -4-methyl-cyclohex- 2-carboxylic acid methyl ester 508 (132 mg, 0.28 mmol) and ( S ) -tetrahydro-furan-3 -Ole 509 (247 mg, 2.8 mmol) was treated with potassium tert -butoxide (251 mg, 2.24 mmol) and heat-sealed at 40 ° C for 16 hours. After cooling, the mixture was treated with 2 M HCl until pH 3, partitioned between ethyl acetate and water, and separated. The organic layer was washed with 5% lithium chloride solution, water, brine, and dried over sodium sulfate. After filtration and concentration, the residue was purified by HPLC using CH ₃ CN (0.1% TFA) / H ₂ O (0.1% TFA) to give 107 mg (70% yield) of Compound 5 as a white powder: MS m / z): 544.0 [M + H] &lt; + &gt;; HPLC retention time 4.22 min (2-98% acetonitrile: water and 0.05% trifluoroacetic acid).

Intermediate compound 508 was prepared as follows.

a. Preparation of Compound 502

To a solution of (S) -3-hydroxy-4,4-dimethyl dihydrofuran-2 (3H) -one (2.60 g, 20 mmol) and diisopropylethylamine (5.2 mL, 30 mmol) in dichloromethane ) Was cooled to -10 [deg.] C and treated dropwise with acryloyl chloride (2.03 mL, 25 mmol) and stirred for 2 h. 1 M HCl (20 mL) was added and the organic layer was washed with sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered, and concentrated. 2.09 g (57% yield) of the desired (S) -4,4-dimethyl-2-oxotetrahydrofuran-3-yl acrylate 501 was obtained as a clear oil by flash chromatography (10-40% EtOAc, hexanes) Respectively.

4-dimethyl-2-oxotetrahydrofuran-3-yl acrylate 501 (2.05 g, 11.1 mmol) in dichloromethane Was treated with titanium tetrachloride (2.2 mL, 1 M in dichloromethane, 2.2 mmol). The yellow solution was stirred for 15 minutes and treated dropwise over 5 minutes with isoprene (1.67 mL, 16.7 mmol). After stirring for 2 hours, an additional portion of isoprene (1.67 mL, 16.7 mmol) was added and the reaction mixture was stirred at -10 to 0 C for 3.5 hours. The reaction mixture was quenched with ammonium chloride (saturated, aqueous). Water and ethyl acetate: hexane (1: 1) were added. The organic layer was separated and the aqueous layer was back extracted with ethyl acetate: hexane (1: 1). The combined organic layers were dried over sodium sulfate, filtered and concentrated. Purification of the residue by flash chromatography (10-50% EtOAc: Hex, 80 g column) afforded 1.30 g (46% yield) of (R) - ((S) -4,4-dimethyl-2-oxotetrahydro Furan-3-yl) 4-methylcyclohex-3-enecarboxylate 502 as a clear oil.

b. Preparation of Compound 503

To a solution of (R) - ((S) -4,4-dimethyl-2-oxotetrahydrofuran-3-yl) 4-methylcyclohexane-2-carboxylate in THF (10 mL), water (1 mL) 3-enecarboxylate 502 (1.30 g, 5.15 mmol) was treated with lithium hydroxide monohydrate (2.16 g, 51.5 mmol) and warmed to 50 &lt; 0 &gt; C with stirring. After 1 h, the reaction mixture was treated with 1M HCl. The mixture was extracted with hexane: THF (10: 1), dried over sodium sulfate, filtered, and concentrated to give 0.738 g (quantitative yield) of (R) -4-methylcyclohex-3-enecarboxylic acid 503 as a white powder Respectively.

c. Preparation of compound 504

(R) -4-Methylcyclohex-3-enecarboxylic acid 503 (371 mg, 2.65 mmol) which had been azeotropically dried by evaporation from toluene was treated with potassium tertiary phosphate (1.13 g, 7.94 mmol) and dichloromethane mL) and treated with dimethylformamide (4 drops). The reaction mixture was cooled to 0 &lt; 0 &gt; C and treated dropwise with oxalyl chloride (0.75 mL, 7.9 mmol). The reaction mixture was allowed to warm to ambient temperature with stirring for 2 hours. After filtering the solid, the solution was concentrated, treated with hexane and concentrated again to give (R) -4-methylcyclohex-3-encarbonyl chloride 504 as a pale yellow oil which was used immediately in the next step.

d. Preparation of compound 506

3-enylcarbonyl chloride 504 (2.65 mmol), 5- (3,3-dimethyl-but-1-ynyl) -3- (1,4- dioxa-spiro [ 4.5] dec-8-ylamino) -thiophene-2-carboxylic acid methyl ester 505 (250 mg, 0.66 mmol) and potassium tertiary phosphate (562 mg, 2.65 mmol) were suspended in dichloroethane Lt; RTI ID = 0.0 &gt; 90 C. &lt; / RTI &gt; After 16 h, the reaction mixture was cooled and partitioned between ethyl acetate and water. The organic layer was separated and the aqueous layer was back extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered and concentrated. Flash chromatography (10-40% EtOAc: hexanes) afforded 220 mg (67% yield) of the desired 5- (3,3-dimethyl-but- 1 -inyl) -3 - [(1,4- [4.5] dec-8-yl) - (( lR ) -4-methyl-cyclohex-3-enecarbonyl) -amino] -thiophene-2-carboxylic acid methyl ester 506 as a beige foam.

e. Preparation of Compound 507

- ((1R) -4-methyl-cyclohex- &lt; / RTI &gt; Carboxylic acid methyl ester 506 (219 mg, 0.438 mmol) was dissolved in THF (3.5 mL) and treated with 4M HCl (1.75 mL, 7.01 mmol). The reaction mixture was heated to 45 &lt; 0 &gt; C and stirred for 2 h. Ethyl acetate was added and the organic layer was then separated and then washed with water, sodium bicarbonate (sat aq), water, and brine. The organic layer was dried over sodium sulfate, filtered and concentrated to give the desired 5-a 0.190 g (95% yield) of (3,3-dimethyl-but-1-ynyl) -3 - [((1 R ) -4- methyl- (4-oxo-cyclohexyl) -amino] -thiophene-2-carboxylic acid methyl ester 507 as a white foam.

f. Preparation of compound 508

Trimethyl sulfoxonium chloride (79 mg, 0.62 mmol) was treated with sodium hydride (21 mg, 60% oil dispersion, 0.53 mmol) in DMSO (1.5 mL) and stirred at ambient temperature for 10 min. (4-methyl-cyclohex-3-enecarbonyl) - (4-methyl- Oxo-cyclohexyl) -amino] -thiophene-2-carboxylic acid methyl ester 507 was added dropwise and the reaction mixture was stirred for 45 minutes. The orange solution was treated with 5% citric acid until pH 3 and partitioned between water and ethyl acetate. The organic layer was separated and the aqueous layer was back extracted with ethyl acetate. The combined organic layers were washed with 5% LiCl, water and brine and dried over sodium sulfate. After filtration and concentration, the residue was purified by flash chromatography (20-75% EtOAc: hexanes) to give 0.134 g (70% yield) of 5- (3,3- dimethyl- ((1 R) -4- methyl-hex-3-bicyclo Enka Viterbo carbonyl) - (1-oxa-spiro [2.5] oct-6-yl) -amino] -thiophene-2-carboxylic acid methyl ester 508 the Obtained as a white powder.

Compound 6 was prepared using synthetic methods and intermediates as described in USSN 12 / 779,023 (US 20100310512 Al). Compound 6 can be prepared as described in the following examples.

Example 6: (1- {3- [6- (9,9-difluoro-7- {2- [5- (2- methoxycarbonylamino-3-methyl-butyryl) -5- -Pyrazol-2-yl) -1H-benzoimidazol-2-yl] -2-aza-bicyclo [3.2.1] hept- 2.2.1] heptane-2-carbonyl} -2-methyl-propyl) -carbamic acid methyl ester 6

Spiro [2.4] hept-6-ene-2-carboxylic acid methyl ester was used in place of 3- [6- (9,9-difluoro-7- {2- [5- Yl] -2-aza-bicyclo [2.2.1] heptane-2-carboxylic acid Butyl ester 614 (115 mg, 0.138 mmol) was dissolved in methylene chloride (2 mL) and HCl in dioxane (4M, 2 mL) was added and stirring was continued at room temperature. After 20 minutes, all volatiles were removed under vacuum. The crude material was used in the next step without further purification. The crude material was dissolved in DMF (1.5 mL) and DIEA (53.4 mg, 0.414 mmol) was added. A solution of 2- ( L ) methoxycarbonylamino-3-methyl-butyric acid 611 (24.2 mg, 0.138 mmol), HATU (52.4 mg, 0.138 mmol) and DIEA (17.8 mg, 0.138 mmol) in DMF (1 mL) . The reaction was stirred at room temperature. After 20 minutes, the reaction was diluted with EtOAc and washed with aqueous bicarbonate solution, aqueous LiCl (5%), brine, and dried over sodium sulfate. Filtered and the solvent removed in vacuo to produce the crude and this RP-HPLC: a (eluent water / MeCN w / 0.1% TFA) to give compound 6 (76 mg) was obtained. LCMS-ESI ⁺ : Calcd for C ₄₉ H ₅₄ F ₂ N ₈ O ₆ : 888.9 (M ⁺ ); Measured: 890.0 (M + H <^+> ).

Intermediate compound 614 was prepared as follows.

a. Preparation of compound 4-methylene-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-methyl ester 602

4-methylene-pyrrolidine-l, 2-dicarboxylic acid l-tert-butyl ester 601 (10.0 g, 44 mmol) was dissolved in MeOH (75 mL) at room temperature and HCl (4M in dioxane, 75 mL) . Stirring at room temperature was continued for 4 hours. All volatiles were removed in vacuo and a beige solid was obtained. The crude material was suspended in methylene chloride (100 mL) and N-methylmorpholine (13.3 g, 132 mmol) was added. The mixture was cooled to 0 C and benzyl chloroformate (8.26 g, 48.4 mmol) was added with stirring. After 30 minutes, the reaction was allowed to warm to room temperature and the solution was washed with water and aqueous HCl (1M). The solution was dried over sodium sulfate. The solvent was filtered and evaporated to yield the crude product which was purified by silica gel chromatography (eluent: EtOAc / hexanes) to give 602 (10.2 g). Calcd for _{C 15 H 17 NO 4::} LCMS-ESI + 275.3 (M +); Measured: 276.4 (M + H <^+> ).

b. Preparation of a mixture of compounds 603 and 604

The oven-dried three neck round bottom flask was equipped with a nitrogen inlet adapter and a 250 mL addition funnel. The third neck was sealed with diaphragm. The flask was charged with stir bar, dichloromethane (120 mL) and diethylzinc (1.0 M in hexane, 118 mL, 118 mmol) and cooled to 0 ° C in an iced ice bath. Dichloromethane (40 mL) and trifluoroacetic acid (9.1 mL, 118 mmol) were added to the addition funnel. After cooling the diethylzinc solution to 0 &lt; 0 &gt; C (about 25 min), the trifluoroacetic acid solution was added dropwise to the stirred reaction mixture over 20 min. After stirring for 20 min at 0 &lt; 0 &gt; C, diiodomethane (9.5 mL, 118 mmol) was added slowly over 4 min. After an additional 20 minutes, 4-methylene-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-methyl ester 602 (8.10 g, 29.4 mmol) was added by cannula to 30 mL dichloromethane. The flask containing 4-methylene-pyrrolidine-1, 2-dicarboxylic acid 1-benzyl ester 2-methyl ester was rinsed with an additional 10 mL of dichloromethane and the solution was also transferred via cannula to the reaction mixture. The reaction mixture was allowed to warm to room temperature and stirred for 110 hours (about 5 days) and then quenched with saturated aqueous ammonium chloride (~ 150 mL). The contents of the flask were slowly poured into a 2 L separatory funnel containing saturated aqueous sodium bicarbonate (800 mL). The aqueous phase was extracted three times with 300 mL ethyl acetate. The combined organic phases were dried over magnesium sulfate and concentrated to give a mixture of compounds 603 and 604 .

c. Preparation of compound 603

The crude material from sub-part b was dissolved in 3: 1: 1 THF / water / acetone (165 mL) and then N -methylmorpholine- N -oxide (3.45 g, 29.4 mmol) and osmium tetraoxide (4 wt% in water, 5 mL, 0.818 mmol). After stirring at room temperature for 7 hours, the reagent was quenched with 1 M aqueous sodium thiosulfate (~ 100 mL). The contents of the flask were then poured into a 1 L separatory funnel containing water (~ 300 mL). The aqueous phase was extracted three times with 300 mL dichloromethane. The combined organics were dried over magnesium sulfate and concentrated. The crude 5-aza-spiro [2.4] heptane-5,6-dicarboxylic acid 5-benzyl ester 6-methyl ester 603 was purified by column chromatography on silica column (5% to 45% EtOAc / hexanes) (5.54 g, 19.15 mmol, 65%).

d. 5-aza-spiro [2.4] heptane-5,6-dicarboxylic acid 5-benzyl ester 606

5-aza-spiro [2.4] heptane-5,6-dicarboxylic acid 5-benzyl ester 6-methyl ester 603 (244 mg, 0.840 mmol) was dissolved in THF (2.0 mL) / MeOH (1.5 mL). An aqueous solution of LiOH (35.5 mg, 0.84 mmol) was added and stirring was continued at room temperature. After 3 h, the reaction was neutralized with aqueous HCl (1 M) and the organic solvent removed in vacuo. The crude mixture was diluted with water and EtOAc and the organic layer was collected. All volatiles were removed in vacuo and the crude acid 606 was used without further purification. Calcd for _{C 15 H 17 NO 4::} LCMS-ESI + 275.3 (M +); Measured: 276.3 (M + H <^+> ).

e. Preparation of 2,7-dibromo-9,9-difluoro-9H-fluorene 608

6-dibromo-fluoren-9-one 607 (4.0 g, 11.8 mmol) was suspended in deoxo fluorine (12 mL) at room temperature and EtOH (4 drops) was added. The stirred suspension was heated at T = 90 占 폚 for 24 hours. ( Note: using deoxo fluorine at elevated temperature as described above may work quickly with care and may cause intense exothermic reaction ). The reaction was cooled to room temperature and poured onto ice containing sodium bicarbonate. A solid formed and was collected by filtration. The crude material was taken up in EtOAc and washed with aqueous HCl (1 M) and brine. The solution was dried over sodium sulfate. Filtration and evaporation of the solvent gave the crude product which was purified by silica gel chromatography (eluent: EtOAc / hexanes) to give compound 608 (3.2 g). ^{19 F-NMR: 282 MHz,} (dmso-d 6) δ: -111.6 ppm. Before using the material in the next step, it was exposed to charcoal as a solution in EtOAc.

f. 5-aza-spiro [2.4] heptane-5,6-dicarboxylic acid 5-benzyl ester To a solution of 6- [2- (7-bromo-9,9-difluoro-9H- -Oxo-ethyl] &lt; / RTI &gt; ester 609

2,7-dibromo-9,9-difluoro--9H- fluorene-608 (372 mg, 1.04 mmol) , Pd (PPh 3) 4 (30.0 mg, 0.026 mmol), PdCl 2 (PPh 3) 2 It was dissolved in (18.2 mg, 0.026 mmol), As (PPh 3) 3 to (5.0 mg) under an atmosphere of argon in dioxane (10 mL). Ethoxyvinyl-tributyltin (376.4 mg, 1.04 mmol) was added. The mixture was heated at 85 占 폚 for 140 minutes (oil bath). The reaction was cooled to room temperature. N -Bromosuccinimide (177 mg, 1.0 mmol) followed by water (2 mL). The reaction was stirred at room temperature for 3 hours, after which most of the dioxane was removed under vacuum. The crude reaction mixture was diluted with EtOAc and washed with water. All volatiles were removed under vacuum. Toluene was added and all volatiles were removed once again under vacuum. The crude material was dissolved in DMF / MeCN (2 mL, 1: 1) at room temperature. A solution of N- CBz-4-cyclopropyl ( L ) proline 606 (0.84 mmol) and DIEA (268 mg, 2.08 mmol) in MeCN (2 mL) was added and stirring was continued at room temperature. After 14 hours, most of the MeCN was removed under vacuum The crude reaction mixture was diluted with EtOAc. The mixture was washed with aqueous HCl (1M), aqueous LiCl solution (5%), brine and dried over sodium sulfate. Filtration and evaporation of the solvent gave the crude reaction product which was purified via silica gel chromatography (eluent: EtOAc / hexanes) to give compound 609 (176 mg). LCMS-ESI ^+: calcd for _{C 30 H 24 BrF 2 NO 5} : 596.4 (M +); Measured: 595.2 / 597.2 (M + H <^+> ).

g. 2-yl] -5-aza-spiro [2.4] heptane-5,6-dicarboxylic acid -Carboxylic acid benzyl ester 610

5-aza-spiro [2.4] heptane-5,6-dicarboxylic acid 5-benzyl ester To a solution of 6- [2- (7-bromo-9,9-difluoro-9H- -Oxo-ethyl] ester 609 (172 mg, 0.293 mmol) was dissolved in m -xylene (6.0 mL). Ammonium acetate (226 mg, 2.93 mmol) was added and the reaction was stirred at 140 &lt; 0 &gt; C for 60 minutes under microwave conditions. The reaction was cooled to room temperature and all volatiles were removed in vacuo. The crude material was purified by silica gel chromatography (eluent: EtOAc / hexane) to give compound 610 (80.3 mg). LCMS-ESI ^+: calcd for _{C 30 H 24 BrF 2 N 3} O 2: 576.4 (M +); Measured: 575.2 / 577.2 (M + H ^& lt ^{; +} & gt ^; ).

h. (L- {6- [5- (7-Bromo-9,9-difluoro-9H-fluoren-2-yl) -lH-imidazol- ] Heptane-5-carbonyl} -2-methyl-propyl) -carbamic acid methyl ester 612

2-yl] -5-aza-spiro [2.4] heptane-5,6-dicarboxylic acid -Carboxylic acid benzyl ester 610 (800 mg, 1.38 mmol) was dissolved in methylene chloride (15 mL) and HBr in AcOH (37%, 2 mL) was added and stirring was continued at room temperature. After 180 min, the suspension was diluted with hexane and the solid was collected by filtration, washed with hexane and vacuum treated. The crude material was used in the next step without further purification. The crude material was dissolved in DMF (4.0 mL) and DIEA (356 mg, 2.76 mmol) was added. DMF (1 mL) of 2- (L) - methoxycarbonyl-amino-3-methyl-butyric acid 611 (242 mg, 1.38 mmol) , HATU (524 mg, 1.38 mmol) and DIEA (178 mg, 1.38 mmol) solution Was added. The reaction was stirred at room temperature. After 50 min, the reaction was diluted with EtOAc and washed with aqueous bicarbonate solution, aqueous LiCl (5%), brine and dried over sodium sulfate. Filtration and removal of the solvent in vacuo yielded the crude material which was purified by silica gel chromatography (eluent: EtOAc / hexane) to give a few impurity compounds 612 (878 mg). LCMS-ESI ⁺ : Calcd for C ₂₉ H ₂₉ BrF ₂ N ₄ O ₃ : 599.5 (M ⁺ ); Measured: 598.5 / 600.5 (M + H ^& lt ^{; +} & gt ^; ).

i. Spiro [2.4] hept-6-ene-2-carboxylic acid methyl ester was used in place of 3- [6- (9,9-difluoro-7- {2- [5- Yl] -2-aza-bicyclo [2.2.1] heptane-2-carboxylic acid Preparation of carboxylic acid tert-butyl ester 614

(L- {6- [5- (7-Bromo-9,9-difluoro-9H-fluoren-2-yl) -lH-imidazol- ] Heptane-5-carbonyl} -2-methyl-propyl) -carbamic acid methyl ester 612 (840 mg, 1.4 mmol), 3- [6- -Benzoimidazol-2-yl] -2-aza-bicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 613 (615 mg, 1.4 mmol), was dissolved in a _{Pd (PPh 3) 4 (161} mg, 0.14 mmol), DME (15 mL) / water (3 mL) under a _{K 2 CO 3 (579 mg,} 4.2 mmol) and argon atmosphere. The mixture was heated at 85-90 &lt; 0 &gt; C (oil bath) for 120 minutes. After 120 min, additional boronate ester (61 mg, 0.14 mmol) was added and heating was continued. After 3 hours, the reaction was cooled to room temperature. Most of the DME is removed under vacuum The crude reaction mixture was diluted with EtOAc. The mixture was washed with brine and dried over sodium sulfate. Filtration and evaporation of the solvent gave the crude reaction product which was purified via silica gel chromatography (eluent: EtOAc / hexanes) to give compound 614 (878 mg). LCMS-ESI ⁺ : Calcd for C ₄₇ H ₅₁ F ₂ N ₇ O ₅ : 831.9 (M ⁺ ); Measured: 832.7 (M + H ^& lt ^{; +} & gt ^; ).

Intermediate compound 613 can be prepared as follows.

j. Preparation of 3- (2-amino-4-bromo-phenylcarbamoyl) -2-aza-bicyclo [2.2.1] heptane-2- carboxylic acid tert -butyl ester 617

To a solution of 2-aza-bicyclo [2.2.1] heptane-2,3-dicarboxylic acid 2- tert -butyl ester 616 (0.327 g, 1.36 mmol, 1 eq.), 4-bromo- HATU (0.543 g, 1.05 eq.) Was added to a solution of 2-diamine 615 (0.507 g, 2.71 mmol, 2 eq.) And 4-methylmorpholine (0.299 mL, 2 eq.). The reaction mixture was stirred at room temperature for 1 hour and then concentrated. The reaction mixture was diluted with ethyl acetate and washed with dilute aqueous NaHCO ₃ and brine. The organic layer was concentrated and purified by flash column chromatography (silica gel, 20-80% ethyl acetate / hexanes) to give the structural isomer 3- (2-amino-4-bromo-phenylcarbamoyl) -2- [2.2.1] heptane-2-carboxylic acid tert -butyl ester 617 was obtained.

k. Preparation of 3- (6-bromo-1H-benzoimidazol-2-yl) -2-aza-bicyclo [2.2.1] heptane-2- carboxylic acid tert -butyl ester 618

Structural isomers of 3- (2-amino-4-bromo-phenylcarbamoyl) -2-aza-bicyclo [2.2.1] heptane-2-carboxylic acid tert-melting and sealing the mixture of the butyl ester in 617 ethanol The tube was heated to 130 &lt; 0 &gt; C overnight and heating was continued at 170 &lt; 0 &gt; C for 3 days. LC-MS showed the desired product and the Boc decomposition product (approximately 1: 1 ratio). The mixture was concentrated and dissolved in HCL. Di- tert -butyl dicarbonate (0.6 eq.) Was added and the reaction was stirred overnight at room temperature. The reaction mixture was concentrated and purified by flash column chromatography (silica gel, 20-80% ethyl acetate / hexanes) to give 3- (6-bromo-1H-benzoimidazol- [2.2.1] heptane-2-carboxylic acid tert -butyl ester 618 (0.383 g, 72%) as an orange foam.

l. Preparation of Compound 613

5 mL of DME 3- (6-bromo -1H- benzoimidazol-2-yl) -2-aza-bicyclo [2.2.1] heptane-2-carboxylic acid tert-butyl ester 618 (264 mg, 0.673 mmol (5 eq., 3.36 g, 6.95 mmol), tetrakis (triphenylphosphine) palladium (5%, 39 mg) and 2M potassium carbonate aqueous solution 3 eq., 1.01 mL) was heated to 90 &lt; 0 &gt; C under Ar for 4 h. The reaction mixture was cooled, diluted with ethyl acetate and washed with saturated sodium bicarbonate solution. The organic layer was dried (MgSO ₄ ), concentrated and purified by flash column chromatography (silica gel, 20-60% ethyl acetate / hexanes) to give 3- {6- [4- (4,4,5,5- [l, 3,2] dioxaborolan-2-yl) -phenyl] -1H- benzoimidazol-2-yl} -2-aza-bicyclo [2.2.1] heptane-2-carboxylic acid tert - Butyl ester 613 (295 mg, 85% yield). _{^{LCMS-ESI -: C 30 H}} 38 BN 3 O 4 Calcd: 515.45; Measured: 516.1 (M + H <^+> ).

Compound 7 could be prepared using synthetic methods and intermediates as described in US 7,429,572. Compound 7 can also be prepared as described in the following examples.

Example 7: Preparation of Compound 7

To an ice-cold suspension of compound 701 (970 g, 3.74 mol) and DMAP (50 g, 0.412 mol) in THF (10 L) was added TEA (2.3 kg, 16.5 mol) and water (7 L) Respectively. Isobutyryl chloride (3 eq.) Was slowly added to the stirred mixture while maintaining a temperature of about 0 &lt; 0 &gt; C. Additional compound 1.2 and then 0.7 equivalents of isobutyl chloride were added until the HPLC essentially indicated that the reaction was complete (about 1.95 kg total). The reaction mixture was acidified with concentrated HCl to a pH of about 6.4 and the organic phase was washed with EtOAc (2 x 10 L). The combined extracts were washed with water (1 x 15 L). The organic phase was filtered and concentrated in vacuo. The residue was dissolved in IPA (about 20 kg) and heptane (14.2 kg) was added. The solution was heated to about 74-75 DEG C to produce a clear solution, and then about 5 L was removed by distillation. The resulting solution was slowly cooled to room temperature. The precipitate was formed at about 42-43 [deg.] C. Cooling was continued to &lt; RTI ID = 0.0 &gt; 5 C &lt; / RTI &gt; The resulting solid was filtered and the filtrate was washed with an IPA / heptane (1: 8) mixture (13.4 kg) and dried under vacuum at about 60-70 ° C to give 1.295 kg (86.65%) of Compound 7 Purity: 99.45%).

Intermediate compound 706 can be prepared as follows.

a. Preparation of compound 701

To the suspension of cytidine (100 g, 0.411 mol) in DMF (2.06 L) was added benzoic anhydride (102.4 g, 0.452 mol). The mixture was stirred at room temperature for 20 hours. The DMF was removed in vacuo and the residue was triturated with diethyl ether. The resulting solid was collected by suction filtration and washed with diethyl ether (2 x 200 mL). Further drying under vacuum at room temperature gave N ⁴ benzamide (140.6 g, 98.3%). A portion of this material (139.3 g, 0.401 mol) was dissolved in anhydrous pyridine (1.2 L) and 1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane (141.4 mL, 0.441 mol) Lt; / RTI > The solution was stirred overnight at room temperature. The mixture was concentrated under near-dry vacuum and co-evaporated with toluene (3 x 200 mL). The residue was treated with EtOAc (1.8 L) and washed with HCl (2 x 200 mL, 0.05 N), NaHCO ₃ (5%, 2 x 400 mL). The organic layer was dried (Na ₂ SO _4), filtered, and evaporated to dryness. Compound 701 (256.5 g, &gt; 100%) was isolated as a white foam and used without further purification.

b. Preparation of compound 702 .

Compound 701 (236.5 g, 0.40 mol) was dissolved in dry THF (1.22 L). Dry DMSO (180.8 mL, 2.1 mol) was added and the resulting solution was cooled to -20 &lt; 0 &gt; C to -15 [deg.] C. Trifluoroacetic anhydride (90.6 mL, 0.64 mol) was added dropwise over 45 minutes and the solution was stirred at -20 &lt; 0 &gt; C to -15 &lt; 0 &gt; C for 2 hours, then anhydrous triethylamine (223.5 mL, 1.6 mol ) Was added over 20 minutes. The crude reaction product containing ketone 702 was dissolved in EtOAc (500 mL) and the resulting solution was washed with H ₂ O (3 x 400 mL), dried (Na ₂ SO ₄ ) and the solvent removed in vacuo to give a yellow solid , Which was purified on a silica gel column eluting with a stepwise gradient (0-60%) of Et ₂ O in hexanes followed by a stepwise gradient (50-100%) of EtOAc in hexanes. The resulting crude ketone (~ 192 g) was crystallized from petroleum ether to give ketone 702 (138.91 g, 57.5% from cytidine) as a white solid and 22 g of unreacted starting material 701 as a yellow solid .

c. Preparation of compound 703

Compound 702 (48.57 g, 8.26 mmol) was dissolved in anhydrous toluene (~ 400 mL) and the solvent was removed from the moisture and removed under vacuum. The residue was then further dried under vacuum (oil pump) for an additional 2 hours. The moisture was strictly excluded and the remaining foam was dissolved in anhydrous diethyl ether (1.03 L) under argon. The resulting solution was cooled to -78 C under argon and MeLi (1.6 M, 258.0 mL, 0.413 mol) was added dropwise via addition funnel. After the addition was complete, the mixture was stirred for 2 h at -78 &lt; 0 &gt; C. Aqueous 1M NH ₄ Cl (500 mL) was slowly added. After warming to room temperature, the mixture was washed with H ₂ O (2 x 500 mL), dried (Na ₂ SO ₄ ) and then concentrated to dryness to give a brown foam (~ 60 g,> 100%).

37.62 g and 56.4 g of compound 702 were used to carry out the reaction two or more times. The combined crude product (128.0 g, 0.212 mol) was dissolved in THF (1.28 L) and treated with concentrated HOAc (23 mL, 0.402 mol). To the solution was added TBAF (384.0 mL, 1 M in THF). The solution was stirred at room temperature for 0.75 h and the mixture was treated with silica gel (750 g) and dried concentrated. The powder was placed on a packed silica gel column in CH ₂ Cl ₂ . A dark waxy solid was obtained by elution with 1: 7 EtOH-CH ₂ Cl ₂ and was pre-adsorbed on silica gel (300 g) and chromatographed as above. Compound 703 (46.4 g, 53.0% from compound 702 ) was isolated as an off-white solid.

d. Preparation of compound 704

Compound 703 (46.0 g, 0.13 mol) was dissolved in anhydrous pyridine and dried under vacuum. The resulting syrup was dissolved in anhydrous pyridine under argon and cooled to 0 &lt; 0 &gt; C with stirring. The brown solution was treated with benzoyl chloride (30 mL, 0.250 mol) dropwise over 10 minutes. The ice bath was removed and stirring continued for 1.5 h, whereby TLC indicated no residual starting material. The mixture was quenched by the addition of water (5 mL) and dried concentrated. The residue was dissolved in a minimal amount of CH ₂ Cl ₂ and washed with saturated NaHCO ₃ (1 x 500 mL) and H ₂ O (1 x 500 mL). The organic phase was dried (Na ₂ SO ₄ ), filtered, dried concentrated and chromatographed on silica gel eluting with an EtOAc-hexane step gradient (25-60%) to give compound 704 as a yellow foam (48.5 g, 67%).

e. Preparation of compound 705

Compound 704 (7.50 g, 0.013 mol) was dissolved in anhydrous toluene (150 mL) under argon and cooled to -20 <0> C. DAST (2.5 mL, 18.9 mmol) was slowly added and the addition was complete and the cooling bath was removed. Stirring was continued for 1 hour, the mixture was poured into saturated NaHCO ₃ (100 mL) and washed until gas evolution be stopped. The organic phase was dried (Na2SO _4), concentrated and 1 was purified by silica gel chromatography eluting with 1 EtOAc- hexane. 1.22 g (16.3%) pure compound 705 was obtained as a white solid. _{mp 241 ℃ (CH 2 Cl 2} - hexane);

Anal. Calcd for C ₃₁ H ₂₆ FN ₃ O ₇ .0.7 H ₂ O Calcd .: C, 63.74; H, 4.72; N, 7.20. Measured: C, 63.71; H, 4.54; N, 7.20.

f. Preparation of compound 706

Compound 705 (6.30 g, 0.011 mol) was suspended in methanolic ammonia (about 7 N, 150 mL) and stirred overnight at room temperature. The solvent was removed in vacuo, co-evaporated with methanol (1 x 20 mL) and pre-adsorbed on silica gel. The white powder was placed on a silica gel column (packed in CHCl ₃ ) and the column eluted with 9% EtOH in CHCl ₃ followed by 17% EtOH and finally 25% EtOH in CHCl ₃ . The fractions containing the product were concentrated, filtered through a 0.4 [mu] m disk and lyophilized from water to give compound 706 , 2.18 g (76%).

_{C 10 H 14 FN 3 O 4} · H ₂ O Calculated analysis of the 1.4: C, 44.22; H, 5.95; N, 14.77. Measured: C, 42.24; H, 5.63; N, 14.54. Compound 706 (0.10 g, 0.386 mmol) was converted to the hydrochloride salt by dissolving in water (2 mL) and the pH was adjusted to approximately 3.0 using 1 M HCl. Water was removed under vacuum and the residue was crystallized from aqueous EtOH to afford 706 as a hydrochloride salt (71.0 mg). mp 243 [deg.] C (dec);

_{^{19 F NMR (DMSO-d 6}} ;): δ 1.69 (m). _{C 10 H 14 FN 3 O 4} · HCl Calculated analysis for: C, 40.62; H, 5.11; N, 14.21. Measured: C, 40.80; H, 5.09; N, 14.23.

Compound 8 was prepared using synthetic methods and intermediates as described in USSN 12 / 632,194. Compound 8 can also be prepared as described in the following examples.

Example 8: Preparation of 4-Amino-2- n -butoxy-8- [3 &apos;-( pyrrolidin- Preparation of 6-one 8 (R = n- butyl)

To a solution of the nitro compound 807 (730 mg, 1.5 mmol) in MeOH (10 mL) Raney nickel (~200 L, slurry in H ₂ O) was added. The reaction tube was stirred for 1.5 h with H ₂ under a H ₂ atmosphere, followed by flushing. The mixture was filtered through Celite using CH ₂ Cl ₂ and MeOH (1: 1). The filtrate was concentrated in vacuo and left on the freeze dryer overnight. The free base of Compound 8 was obtained as a white solid. To obtain the HCl salt of Compound 8, the above filtrate sample was spiked with 1.0 M HCl to pH = 1-2 and lyophilized.

_{^{LCMS-ESI +: C 22 H}} 31 N 6 O Calculated for ^{_{2: 411.5 (M + H +}} ); Measured: 411.3 (M + H <^+> ).

Intermediate compound 807 was prepared as follows.

a. Preparation of compound 802

At -20 ℃ To a solution of 801 (2.46 g, 10.2 mmol) in _{THF (34 mL), Et 3} N was added (3.14 mL, 22.5 mmol), and then NH ₃ (MeOH of 2.0 M, 5.4 mL, 11 mmol ) Was added. The mixture was stirred for 1.5 h while warming to 0 &lt; 0 &gt; C (LC / MS indicated consumption of starting material). The reaction mixture containing compound 802 was collected without workup.

b. Preparation of compound 803

To a solution of 3 - ((1-pyrrolidinylmethyl) phenyl) methanamine 806 (1.95 g, 10.2 mmol) in THF (34 mL) at 0 ° C was added Et ₃ N (3.14 mmol, 22.5 mmol) followed by methyl bromo Acetate (1.04 mL, 22.3 mmol) was added dropwise. The reaction mixture was stirred for approximately 2 hours until LC / MS indicated that the starting material was consumed. A mixture containing compound 803 was taken for work-up.

c. Preparation of compound 804

Compound 803 Was added to the reaction mixture at &lt; RTI ID = 0.0 &gt; 0 C &lt; / RTI &gt; The reaction mixture was stirred for approximately 45 minutes until LC / MS indicated consumption of compound 802 . A saturated solution of NH ₄ Cl (50 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (2 x 30 mL). Dry the combined organic layers over MgSO _4, filtered, and concentrated in vacuo. Purification by silica gel chromatography gave 2.11 g of compound 804 .

_{^{LCMS-ESI +: C 21 H}} 29 N 6 O calcd for _{4 S: 461.2 (M + H} +); Measured: 461.0 (M + H <^+> ).

d. Ethyl _α -N [4- amino-2-methanesulfonyl-5-you trophy _{limiter-6-yl], N α [3 '-} ( pyrrolidine-1 "-yl-methyl) -benzyl] glycinate Manufacture of Nate 805

To a suspension of Sulfide 804 (3.68 g, 8.00 mmol) in EtOH (40 mL) at 0 C was added sodium tungstate dihydrate (792 mg, 2.40 mmol), acetic acid (4.6 mL, 80 mmol), and hydrogen peroxide (3.4 mL, ~ 40 mmol, 35% w / w in H ₂ O) were added sequentially. After 3 hours, additional acetic acid (4.6 mL) and hydrogen peroxide (3.4 mL) were added. The reaction was held at 0 &lt; 0 &gt; C for 16 hours. A saturated solution of Na ₂ SO ₃ (50 mL) was carefully added at 0 ° C and then CH ₂ Cl ₂ (75 mL) was added. The layers were separated and the aqueous layer was extracted with CH ₂ Cl ₂ (4 x 50 mL). The combined organic layers were dried over MgSO _4, filtered, and concentrated under vacuum to give a material containing a compound 805 without further purification.

e. Preparation of compound 807 (R = n-butyl)

To a solution of sulfone 805 (1.0 g, 2.0 mmol) in n-butanol (10 mL) was added TFA (470 L, 6.1 mmol). The reaction was stirred at 100 &lt; 0 &gt; C for 1 hour. The reaction mixture was poured into a saturated solution of NaHCO ₃ (20 mL) and CH ₂ Cl ₂ (30 mL). The layers were separated and the aqueous layer was extracted with CH ₂ Cl ₂ (30 mL). Dry the combined organic layers over MgSO _4, filtered, and concentrated in vacuo. By silica gel chromatography (1 g matrix / 10 g SiO ₂₎ compound 807 by performing the purification _{(2-15% MeOH / CH 2 Cl} 2) was obtained.

Example 9 : Preparation of Compound 9 (US2010 / 0298257)

(S) -2 - {[(1R, 4R, 5R) -5- (2,4-Dioxo-3,4-dihydro-2H-pyrimidin- Methyl-tetrahydro-furan-2-yl-methoxy] -phenoxyphosphorylamino} -propionic acid isopropyl ester (US2010 / 0298257 Example 2)

Synonyms: 5'-O- (isopropyl-L-alanate, phenylphosphoramidyl) -2'-deoxy-2'-fluoro-2'-C-methyl-uridine diastereomeric mixture.

A 5 L three-necked flask equipped with a mechanical stirrer, brine ice bath, internal thermometer and nitrogen atmosphere was used. The flask was charged with L-alanine isopropyl ester hydrogen chloride (82.0 g, 0.490 mol) and anhydrous dichloromethane (0.80 L). While stirring, phenyldichlorophosphate (85.0 g, 0.40 mol) was added in one portion and stirred. A solution of N-methylimidazole (NMI, 250 g, 3.07 mol) in dichloromethane (250 mL) was added over 30 minutes while maintaining the internal temperature at -5 to 5 占 폚. The solution was stirred at this temperature range for 1 hour. 2'-fluoro-2'-C-methyl-uridine (3,80.0 g, 0.307 mol) was added in one portion at 0 ° C and the reaction flask was then slowly warmed in a salt bath. After 1 hour, the internal temperature was raised to -2 [deg.] C. At 1 h TLC (5% methanol in HCL) showed that more than 50% of the nucleoside was consumed. The bath was removed and the reaction flask reached ambient temperature over 1 h. After 3 h and 5 h total TLC indicated that 95% of the starting nucleoside was consumed. The reaction mixture was quenched by the addition of methanol (100 mL) and the reaction was stirred for 5 min.

The reaction mixture was washed with 1N HCl (2x500 mL) followed by saturated sodium bicarbonate solution (2x500 mL). The separated organic layer was dried over anhydrous sodium sulfate (50 g) and filtered. The solution was evaporated under reduced pressure and then evaporated under high vacuum to give the crude product as a viscous oil (170 g). NMR of the crude product ( ³¹ P and ¹ H) was taken. ³¹ P-NMR showed about 1% of total phosphorus incorporation due to the presence of 3 'isomer 5.

The crude product was added to anhydrous pyridine (1700 mL). The solvent was evaporated under reduced pressure and then evaporated under high vacuum to reduce the moisture content of the crude mixture through co-evaporation. The oil was re-dissolved in anhydrous pyridine (500 ml) and then excess t-butyldimethylsilyl chloride (9.0 g, 60 mM) was added. The reaction was stirred at ambient temperature. The progress of the reaction was monitored by UPLC / MS. After 3 hours, the 3 'impurity 5 could no longer be detected and the reaction was quenched by the addition of methanol (50 mL).

The reaction was evaporated under reduced pressure to give an oil. The residue was dissolved in ethyl acetate (1.5 L), washed with 1 N HCl (2 x 500 mL), and then with saturated sodium bicarbonate solution (2 x 500 mL). The organic layer was dried over anhydrous sodium sulfate (50 g), filtered and evaporated under reduced pressure to give the crude product as a pale yellow oil.

The crude oil was diluted to the same volume as dichloromethane and loaded on a 2.5 Kg silica gel cartridge n (radial compression factor at 100 psi air pressure). A 60 psi gradient pump was used and a flow rate of 400 ml / min was used, and the cartridge was washed with methylene chloride (4 L) and washed with methylene chloride (48 L) with gradient 1-4% methanol. Most of the impurities (di- (isopropylalanyl) phenyl phosphate, 3 ', 5'-bisphosphoramidate, 3'-phosphoramidate-5'-TBDMS adduct (7) Respectively. The desired product was eluted with 3-4% methanol. The product containing the fraction was divided into two parts. The first part contained a small amount of the upper impurity and the second part was the pure product. The first set of fractions contained small amounts of less polar impurities (upper impurity) such as 3 ', 5'-bisphosphoramidate and di-alanyl phenyl phosphate and contained most of the Rp diastereomer and the second column purification . (The relative terms top to bottom refer to the eluent on silica-gel chromatography, where the "upper isomer" refers to the first eluting isomer.) The second set of fractions does not have a large amount of impurities - What is present is Rp and most Sp diastereomers. This is then recombined with the columnar fraction two times. The solvent was evaporated under reduced pressure and the resulting white foam was further dried (0.20 mm Hg) for 1 h to give 42 g of impurity portion (4: 1 upper vs. lower isomer based on ³¹ P-NMR) and 38 g pure portion (1: 3 top to bottom isomer). The impurity portion was recolumnzed in a similar manner to yield 3.8 g of 97% pure upper isomer (4: 1 ratio of fraction removed and 36 g pure product). The two major fractions were dissolved in HCL, combined and evaporated under reduced pressure and dried (50 C, 0.2 mm Hg, 24 h) to give 74 g (45.7%) of pure product (Compound 9) as a white foam with 48: &Lt; / RTI &gt; ratio of diastereomers. mp about 75-85 [deg.] C.

To prepare an amorphous solid of the diastereomeric mixture, 74 g of a white foam was stirred with t-butyl methyl ether (750 mL) to give a viscous solid residue as the partial solution. With stirring, heptane (750 mL) was added slowly and mechanically stirred for 1 hour until most of the gum was converted to a white solid. The solid was rubbed with spatula and the resulting slurry was filtered. The solid was washed with heptane (4 x 50 mL) and dried under vacuum (50 캜, 0.2 mm Hg, 24 h) to yield a white amorphous powder (64 g) with a broad melting range of about 70-80 캜. ^The structure was matched by ¹ H and ³¹ P NMR and HPLC showed 99.8% purity with a diastereomer at a ratio of 46:54 (confirmed by ³¹ P NMR).

A solid mixture of Compound 9 was prepared in an alternate manner. After chromatography, the residue was co-evaporated twice with dichloromethane (5 mL / g) and dried at 35-40 [deg.] C and 35-45 mTorr for 24 h. The foam residue was sieved through a 250 micron screen and further dried under the same conditions until the remaining dichloromethane fell below 400 ppm as measured by headspace GC. The fine product of the off-white to white amorphous powder has a glass transition temperature of 53.7 to 63.5 캜.

Characterization of Compound 9 (isomer mixture):

³¹ P-NMR (CDCl ₃ ) 03.60 (Rp) for triphenyl phosphate at -17.80 ppm , 3.20 Sp. ES-MS M + 1 530.2. Component analysis:% calculated (including 0.29% water, Karl Fisher assay) C, 49.75; H, 5.54; N, 7.90, F, 3.58, P, 5.84. Measured%: C, 49.50; H, 5.44; N, 7.85; F, 3.62; P, 6.05.

Preparation of 2'-deoxy-2'-fluoro-2'-C-methyluridine (US2010 / 0298257 Example 1)

To a 10 L flask was added 3'5'-O-dibenzoyl-2'-deoxy-2'-fluoro-2'-C-methyl-N4-benzoylcytidine (500 g, 0.874 mol) Acetic acid (7.5 L) was added. The solution was heated to reflux (110 &lt; 0 &gt; C) for 20 h. TLC showed the reaction was complete (RfO.6 in 5% methanol in dichloromethane (HCL)). The mixture was cooled to ambient temperature and diluted with water (2 L). After stirring for 2 hours, the resulting precipitate was collected by filtration, and the solid was rinsed with water (5 L) and dried in an ambient temperature atmosphere for 12 hours to give 360 g (88%). All of these dibenzoyl uridine intermediates were used directly in the next step by addition to freshly prepared methanolic ammonia (5.4 L, ~ 25%) at 0 ° C. This temperature was maintained for 3 hours and then allowed to warm to 15 DEG C for 24 hours. TLC indicated the reaction was complete (Rf 0.4 at 10% methanol in HCL). The reaction mixture was filtered through a celite layer and concentrated under reduced pressure to give the crude product (216 g). The crude product was stirred for 3 h at ambient temperature with ethyl acetate (325 mL). The resulting solid was collected by filtration and washed with ethyl acetate (216 mL). The solid was dried under vacuum at ambient temperature for 4 hours to give 160 g (78%) of the desired product in 98.7% HPLC purity.

Example 10 : Preparation of compound 10 (US2010 / 0298257)

Direct precipitation of compound 10 (US2010 / 0298257; Example 4): To a solution of 10 g of L-alanine isopropyl ester hydrochloride (10.5 g, 61.5 mmol, azeotropically dry, twice, each time using 50 mL of toluene) in dichloromethane To the stirring solution, at room temperature was added phenyldichlorophosphate (7.5 mL, 50 mmol). The mixture was cooled to -10 C and then a solution of N-methylimidazole (30.5 mL, 384.3 mmol) in 30 mL of dichloromethane was added over 30 minutes. After the addition was complete, the mixture was stirred at -10 to -15 [deg.] C for 1 hour. To the mixture was added in one portion 2'-deoxy-2'-fluoro-2'-C-methyluridine (10 g, 38.4 mmol) (see US2010 / 0298257 Example 1) Lt; 0 &gt; C and then warmed to 20 &lt; 0 &gt; C (6 h). The mixture was stirred at this temperature overnight (15 h) and then quenched with 10 mL of methanol. The solvent was evaporated and the residue redissolved in EtOAc (200 mL). The EtOAc layer was washed with water (100 mL), 1N HCl (3 x 75 mL), 2% aqueous NaHCO ₃ solution (50 mL) and brine (50 mL). The organic layer was dried over Na ₂ SO _4, filtered and concentrated. The residue was dried under high vacuum for 2 hours to give a white foam (22 g).

The foam was dissolved in 33 mL of HCl and then 65 mL of isopropyl ether was added to give a saturated solution. The solution was filtered through a small pad of celite and the filtrate was cooled to ambient temperature (approximately 22 [deg.] C for 72 h using a seed of compound 10 at 0 [deg.] C resulting in oily product free crude product ). The white solid was filtered and washed with isopropyl ether (20 mL) to give a white powder of 4.58 g (~ 85: 15 mixture of compound 10: R isomer (at P each as determined by ³¹ P NMR)) . The solid was suspended in 23 mL of HCl and then refluxed for 3 hours. The mixture was cooled to room temperature and stirred for 15 hours. The white solid was filtered off, washed with 4.5 mL of cold HCl and dried at 45 &lt; 0 &gt; C under high vacuum to yield pure compound 10. mp 93.9-104.7 [deg.] C. HPLC purity 99.74% (3.11 g, 15.2% from uridine nucleoside).

Compound 10:

Example 11: Preparation of compound 11 (US 2010/0081628)

6-Ethoxy-9- ((4aR, 6R, 7R, 7aR) -7-Fluoro-2-isopropoxy- d] [1,3,2] dioxaphosphinin-6-yl) -9H-purin-2-yl-amine (Compound 11) (Compound 19, US 2010/0081628)

(2R, 3R, 4R, 5R) -5- (2-Amino-6-ethoxy-purin-9- yl) -4-fluoro-2-hydroxymethyl- -Ol (150 mg, 0.46 mmol) was dissolved in anhydrous pyridine (2 ml) at 0 &lt; 0 &gt; C. A solution of 0.45 M 1H-tetrazole in acetonitrile (2.55 mL) was added followed by bis (N, N-diisopropylamino) isopropylphosphoramidite (0.16 mL, 0.55 mmol, 1.2 eq). The mixture was slowly warmed to ambient temperature over 3 hours. TLC indicated the reaction was complete. The reaction was quenched by the addition of water (0.1 mL). The reaction solution was concentrated under reduced pressure and the residue was triturated with ethyl acetate (5 mL). The resulting white precipitate was removed by filtration and the filtrate was concentrated under reduced pressure.

The resulting intermediate cyclic phosphite residue was dissolved in acetonitrile (2 mL) and treated with t-butyl hydroperoxide (70% in water, 0.19 mL) at ambient temperature for 5 hours. TLC indicated the reaction was complete. The reaction solution was concentrated under reduced pressure and the residue was purified by column chromatography (Analogix, using 0-5% gradient IPA in HCL). Two diastereoisomers (compound 11 and R-isomer (in P)) could be separated. The fractions containing each diastereomer were separated and combined and concentrated under reduced pressure to a white solid to give 20 mg of each diastereomer (combined yield 20%).

Compound 11

The R-isomer (in P)

The structures for compound 11 and the R-isomer (in P) are shown below.

(2R, 3R, 4R, 5R) -5- (2-Amino-6-ethoxy-9H-purin-9- Furan-3-ol (Compound 16, US 2010/0081628)

To a 500 mL dry round bottom flask was added a solution of (2R, 3R, 4R, 5R) -5- (2-amino-6-chloro-9H- purin- -4-methyltetrahydrofuran-3-ylbenzoate (11 g, 20.92 mmol) was loaded. Anhydrous ethanol (210 mL) was added followed by anhydrous K ₂ CO ₃ (28.91 g, 209.2 mmol). The suspension was stirred and heated at 75 [deg.] C under nitrogen for 5.5 hours. The TLC test confirmed that all starting materials were consumed. The mixture was cooled to room temperature and the solid was filtered off. The filtrate was neutralized to pH ~ 7 by the addition of glacial acetic acid (2.52 g) and concentrated under reduced pressure. The residue was dissolved in methanol and mixed with silica gel (15 g). The dry mixture of crude product and the silica gel were transferred to an empty cartridge and separated by column chromatography (Analogix 220 g, 0-15% MeOH in gradient DCM) to give the product (5% MeOH in DCM) as a white foam 3.73 g, 54.5%). The second white solid was isolated from the column (10% MeOH in DCM, 1.44 g), which is a mixture of two dimers of the nucleoside. A third, more polar white solid was collected from the column (15% MeOH in DCM, 0.47 g), which was a mixture of trimesters of nucleosides. The HPLC purity of the product was 99.94%.

(2-amino-6-chloro-9H-purin-9-yl) -2- Synthesis of 3-yl benzoate (Compound 12, US 2010/0081628)

A 12 L three neck round bottom flask was charged with 6-chloro-2-aminopurine (225.4 g, 1.329 mol). Anhydrous tert-BuOH (4.5 L) was added and the solution was stirred at ambient temperature using a mechanical stirrer. Potassium tert-butoxide (solid, 151.6 g, 1.35 mol) was added in portions under nitrogen gas flow with stirring. The mixture was stirred at room temperature for a further 30 minutes. A 5-L round bottom flask was loaded with alpha -bromide (10, 197 g, 0.451 mol) and 3 L of anhydrous acetonitrile at ambient temperature. The bromide solution was added to the purine base suspension at ambient temperature over one minute. The 5 L flask was rinsed with acetonitrile (2 x 1 L) and the bromide was completely transferred to the reaction mixture. The mixture was heated gradually to 50 &lt; 0 &gt; C over 2 h using a heating mantle and a controller and stirred for 20 h. The reaction was almost complete by TLC beta (R _f 0.28, 30% EtOAc in hexanes). The reaction was quenched by addition of saturated NH ₄ Cl (200 mL) to form a suspension. The suspended solids were removed by filtration through a Celite pad (3 cm) in a 2.5 L porcelain Buchner funnel. The solids were washed with toluene (3 x 100 mL). The combined filtrates were neutralized by the addition of 6 N HCl solution (approximately 220 mL) until the pH was 7. The mixture was concentrated under reduced pressure. When the volume of the mixture was reduced to about 1/3 volume, additional precipitated solids were filtered off in a similar manner. The filtrate was further concentrated to a volume of about 800 mL. The residue was loaded onto a plug column (1.6 kg flash grade silica gel in a 6 L sintered glass glass Buchner funnel) and eluted with 10% gradient ethyl acetate in hexanes (6 L) to remove nonpolar impurities Removed and eluted with 30% ethyl acetate in hexanes to give a small amount of lactol (6 L) followed by the elution of large amounts of product using 40% -45% ethyl acetate in hexanes (4 L). The product containing fractions were combined and concentrated under reduced pressure and dried under vacuum (0.2 mm Hg, 24 h, ambient temperature) to give a white foam solid (150.7 g, b / α = 14: 1 by NMR). ¹ H-NMR. (CDCl ₃₎ beta: δ 1.33 (d, 22.4 Hz , 2'-C-CH 3), alpha: 1.55 (d, 22 Hz, 2'-C-CHH 3).

The product mixture foam was dissolved in methanol (700 mL) at ambient temperature. Upon standing, solid formed slowly over 2 hours. The suspension was cooled to -5 [deg.] C in a freezer for 17 hours. The resulting white solid was collected by filtration and washed with cold MeOH (-5 [deg.] C, 3x60 mL) and ethyl ether (3x100 mL). The solid was dried in vacuo (0.2 mm Hg, 24 h, ambient temperature) to give 110.5 g of [beta] -product ([beta] / [alpha] 99.8: 1 by HPLC). The filtrate was partially concentrated (about 400 mL) and diluted with more MeOH (400 mL) while heating to 60 &lt; 0 &gt; C. The solution was cooled to ambient temperature, seeded and cooled to -5 [deg.] C. The second workpiece was collected, washed and dried in a similar manner to yield a greater amount of product as a white solid (12.26 g) (similar diastereomeric purity). The mother liquor was dried and concentrated under reduced pressure (about 25 g). The residue was a mixture of? And? -Isomers. This was treated with automated silica gel column chromatography (Analogix, 240 g cartridge, 40% to 50% ethyl acetate in hexanes) to yield 14.52 g of product foam which was recrystallized from MeOH, washed and dried in a similar manner To give an additional 8.46 g of product in high purity.

The three solids were judged to be of similar purity and were combined to give 131.2 g of a white crystalline product (55% from bromo sugar and 49% from lactol). Mp 160.5-162.0 DEG C HPLC purity 99.0% including 0.20% alpha.

Example 12: Preparation of compound 12 (US20110015146)

(2S, 3R, 4R, 5R) -5 (2-amino-6-methoxy-9H-purin- Methyl tetrahydrofuran-2-yl) methoxy) (phenoxy) phospholylamino) propanoate Synthesis of

Phenyldichlorophosphate (2.66 g, 12.61 mmol) and anhydrous dichloromethane (40 mL) were loaded into a 250 mL dry round bottom flask. The amino ester salt (2.60 g, 15.53 mmol) was added to the solution and the mixture was cooled to -5 &lt; 0 &gt; C. Then, N-methyl imidazole (7.7 mL, 97 mmol) was added rapidly at -5 DEG C through a dry syringe and the solution was stirred at -5 DEG C for 1 hour. (2R, 3R, 4R, 5R) -5- (2-Amino-6-methoxy-9H-purin-9- Methyl tetrahydrofuran-3-ol), 3.04 g, 9.7 mmol) was added in one portion at -5 [deg.] C from the vial and the solids were slowly dissolved for 20 minutes. The reaction temperature was raised to ambient temperature over 2 hours. After 17 hours, the reaction was not complete. More reagents were prepared and added to the reaction mixture at -5 ° C (from phosphate (2.66 g), amino ester (2.60 g), and N-methyl imidazole (3.8 mL, 48 mmol). The reaction was stirred at room temperature for 2 hours or more. TLC showed the reaction was almost complete and was diluted with 70 mL of dichloromethane. HCI solution (1 N, 70 mL) was added. The aqueous layer was separated and extracted with dichloromethane. The organic layer was washed with saturated NaHCO ₃ , water, brine and dried over MgSO ₄ . After removing the solvent under reduced pressure, the sticky residue was purified by automated column chromatography using 240 g of cartridge and 0-8% gradient 2-PrOH in dichloromethane to give the product as a foam solid (4.16 g, 7.14 mmol, 73% yield). HPLC purity 97.4%. NMR spectrum of the product showed it to be two stereoisomers with a ratio of 1.2: 1.

^{1 H-NMR (DMSO-d} 6): δ 7.98 (1H, s, 8-H of one isomer), 7.95 (1H, s, 8-H addition of the other isomer), 7.37-7.32 (2H, m, arom-H), 7.22-7.15 (3H , m, arom-H), 6.6 (2H, s, NH 2), 6.11 (1H, d, Cl'-H) of the single isomer, 6.09 (1H, d, (1H, d, 3'-OH in one isomer), 5.81 (1H, d, 3'-OH in one isomer) (2H, m, C4'-H, H in the amino ester), 4.15-4.07 (1H, m, C3'- H), 3.96 (3H, s , OCH 3), 3.82-3.72 (2H, m, C5'-H a , and _{C5'H b), 1.23-1.06 (9H,} m, of the amino ester _{CH 3 '), 1.03 (3H} , d, C2'-CH 3).

³¹ P-NMR (DMSO-d6): 0 = 4.91 (one isomer), 4.72 (another isomer).

An alternative purification method is to chemically alter small amounts of 3 ' phosphoramidate byproducts to simplify chromatographic separation. The crude phosphorphoramidate product was dissolved in anhydrous pyridine (5 mL / g) and 0.5 molar equivalents of t-butyldimethylsilyl chloride was treated at ambient temperature to select 3 'isomeric impurities of the free 5' major hydroxy L Lt; / RTI &gt; The progress of the reaction was monitored by LC / MS. Once the 3 'isomer is converted to the 5'-tBDMS-3'-phosphoramidate derivative, the reaction is quenched with methanol (3 eq), concentrated under reduced pressure, partitioned between ethyl acetate and 5% citric acid, The layers were concentrated. Thereafter, the residue was chromatographed and treated with higher loading and rapid gradient to achieve higher purity.

Example 13: Preparation of compound 13 (US20110015146)

Example 14: Preparation of Compound 14 (US 7,964,580, Example 5)

2'-deoxy-2'-fluoro-2'-C-methyluridine-5'-phenylmethoxy-alanylphosphate)

(1 g, 6.5 eq) dissolved in 3 mL of THF was added dropwise to a solution of 2'-deoxy-2'-fluoro-2'-fluoro-2'- Was added to a mixture of C-methyluridine (0.15 g, 1 eq) and N-methylimidazole (0.3 g, 8 eq) and the reaction was then stirred overnight. The solvent was removed under reduced pressure. The resulting crude product was purified by pre-HPLC on a YMC 25x30x2 mm column using a water / acetonitrile gradient elution mobile phase. The acetonitrile and water were removed under reduced pressure to give the desired product (50.1 mg, 15.6%).

Example 15: Preparation of compound 15 (Example 55, US 7,964, 580)

A general procedure for nucleoside phosphoramidate derivatives is reported in US Pat. No. 7,964,580, column 461. A solution of the appropriate phosphorochloridate (6.5 eq.) In anhydrous tetrahydrofuran (THF) was added to a solution of N-methylimidazole (8 eq.) And a nucleophile in anhydrous THF 0.0 &gt; (1 &lt; / RTI &gt; equivalent). The solvent can be removed in vacuo, the crude can be purified by column chromatography and purified by preparative thin layer chromatography to give the desired compound.

Example 16: Preparation of compound 16 (US 7,429,572)

Synthesis of (2'R) -2'-deoxy-2'-fluoro-2'-C-methylcytidine starting from cytidine

Step 1: To a suspension of cytidine (100 g, 0.411 mol) in DMF (2.06 L) was added benzoic anhydride (102.4 g, 0.452 mol). The mixture was stirred at room temperature for 20 h. The DMF was removed in vacuo and the residue was triturated with diethyl ether. The resulting solid was collected by suction filtration and washed with diethyl ether (2 x 200 mL). Further drying at room temperature under vacuum provided N ⁴ benzamide (140.6 g, 98.3%). A portion of this material (139.3 g, 0.401 mol) was dissolved in anhydrous pyridine (1.2 L) and 1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane (141.4 mL, 0.441 mol) Lt; / RTI > The solution was stirred overnight at room temperature. The mixture was concentrated in vacuo to dryness and co-evaporated with toluene (3 x 200 mL). The residue was treated with EtOAc (1.8 L) and treated with HCl (2 x 200 mL, 0.05 N), NaHCO ₃ (5%, 2 x 400 mL). The organic layer was washed, dried (Na ₂ SO ₄ ), filtered and evaporated to dryness. Compound 16-1 (Compound 4-1, US 7,429,572) (256.5 g, &gt; 100%) was isolated as a white foam and used without further purification.

Step 2: Compound 16-1 (236.5 g, 0.40 mol) was dissolved in dry THF (1.22 L). Dry DMSO (180.8 mL, 2.1 mol) was added and the resulting solution was cooled to -20 &lt; 0 &gt; C to -15 [deg.] C. Trifluoroacetic anhydride (90.6 mL, 0.64 mol) was added dropwise over 45 minutes and the solution was stirred for 2 hours at -20 [deg.] C to -15 [deg.] C, then anhydrous triethylamine (223.5 mL, 1.6 mol ) Was added over 20 minutes. The crude reaction product containing ketone 16-2 was dissolved in EtOAc (500 mL) and the resulting solution was washed with H ₂ O (3 x 400 mL), dried (Na ₂ SO ₄ ) and the solvent removed in vacuo to give a yellow solid Which was purified on a silica gel column eluting with a stepwise gradient Et ₂ O (0-60%) in hexanes followed by a stepwise gradient of EtOAc in hexanes (50-100%). The resulting crude ketone (~ 192 g) was crystallized from petroleum ether to give ketone 16-2 (compound 4-2, US 7,429,572) (138.91 g, 57.5% from cytidine) as a white solid and 22 g of Unreacted starting material, 16-1, was obtained as a yellow solid.

Step 3: Compound 16-2 (48.57 g, 8.26 mmol) was dissolved in anhydrous toluene (~ 400 mL) and the solvent was removed under vacuum with the exception of moisture. The residue was then further dried under vacuum (oil pump) for an additional 2 hours. The moisture was strictly excluded and the remaining foam was dissolved in anhydrous diethyl ether (1.03 L) under argon. The resulting solution was cooled to -78 C under argon and MeLi (1.6 M, 258.0 mL, 0.413 mol) was added dropwise via addition funnel. After the addition was complete, the mixture was stirred for 2 h at -78 &lt; 0 &gt; C. Aqueous 1M NH ₄ Cl (500 mL) was slowly added. After warming to room temperature, the mixture was washed with H ₂ O ( ₂ x 500 mL), dried (Na ₂ SO ₄ ) and concentrated to dryness to give a brown foam (~ 60 g,> 100%). 37.62 g and 56.4 g of the compound 16-2 were used to carry out the reaction twice or more. The combined crude product (128.0 g, 0.212 mol) was dissolved in THF (1.28 L) and treated with concentrated HOAc (23 mL, 0.402 mol). To the solution was added TBAF (384.0 mL, 1 M in THF). The solution was stirred at room temperature for 0.75 h and the mixture was treated with silica gel (750 g) and dried concentrated. The powder was placed in a packed silica gel column in CH ₂ Cl ₂ . Pre-adsorbed thick wax solids on silica gel (300 g) eluting with 1: 7 EtOH-CH ₂ Cl ₂ were obtained and chromatographed as above. Compound 16-3 (compound 4-3, US 7,429,572) (46.4 g, 53.0% from compound 16-2) was isolated as an off-white solid.

Anal. Calcd for C ₁₇ H ₁₉ N ₃ O ₆ .0.5 H ₂ O Calculated: C, 55.13; H, 5.44; N, 11.35. Measured: C, 55.21; H, 5.47; N, 11.33.

Step 4: Compound 16-3 (46.0 g, 0.13 mol) was dissolved in anhydrous pyridine and concentrated to dryness under vacuum. The resulting syrup was dissolved in anhydrous pyridine under argon and cooled to 0 &lt; 0 &gt; C with stirring. The brown solution was treated with benzoyl chloride (30 mL, 0.250 mol) dropwise over 10 minutes. The ice bath was removed and stirring continued for 1.5 h, whereby TLC indicated no residual starting material. The mixture was quenched by addition of water (5 mL) and dried concentrated. The residue was dissolved in a minimal amount of CH ₂ Cl ₂ and washed with saturated NaHCO ₃ (1 × 500 mL) and H ₂ O (1 × 500 mL). The organic phase was dried (Na ₂ SO ₄ ), filtered, dried concentrated and chromatographed on silica gel eluting with a stepwise gradient of EtOAc-hexanes (25-60%) to give compound 16-4 as a yellow foam Compound 4-4, US 7,429,572) (48.5 g, 67%).

Measured: C, 65.59; H, 4.79; N, 7.16.

Step 5: Compound 16-4 (7.50 g, 0.013 mol) was dissolved in anhydrous toluene (150 mL) under argon and cooled to -20 <0> C. DAST (2.5 mL, 18.9 mmol) was added slowly and the cooling bath was removed after addition.

Stirring was continued for 1 hour and the mixture was poured into saturated NaHCO ₃ (100 mL) and washed until the gas evolution ceased. The organic phase was dried (Na ₂ SO _4), concentrated, 1: 1, eluting with EtOAc- hexane and purified by silica gel chromatography. 1.22 g (16.3%) of pure compound 16-5 (compound 4-5, US 7,429,572) was obtained as a white solid. _{mp 241 ℃ (CH 2 Cl 2} - hexane);

¹⁹ F NMR (CDCl ₃ ):? 3.3 (m). Anal. Calcd for C ₃₁ H ₂₆ FN ₃ O ₇ .O.7 H ₂ O: C, 63.74; H, 4.72; N, 7.20. Measured: C, 63.71; H, 4.54; N, 7.20.

Step 6: Compound 16-5 (6.30 g, 0.011 mol) was suspended in methanolic ammonia (about 7 N, 150 mnL) and stirred overnight at room temperature. The solvent was removed in vacuo, co-evaporated with methanol (1 x 20 mL) and pre-adsorbed onto silica gel. The white powder was placed on a silica gel column (packed in CHCl ₃ ) and the column was eluted with 9% EtOH followed by elution with 17% EtOH and finally 25% EtOH (in CHCl ₃ ).

The fractions containing the product were concentrated, filtered through a 0.4 [mu] m disk and lyophilized from water to give 2.16 g (76%) of compound 16-6 (compound 4-6, US 7,429,572).

_{^{19 F NMR (DMSO-d 6}} ): δ2.60 (m). C ₁₀ H ₁₄ FN ₃ O ₄ .4.1.4 Alternate Analysis to H ₂ O Calculated: C, 44.22; H, 5.95; N, 14.77. Measured: C, 42.24; H, 5.63; N, 14.54. Compound 16-6 (0.10 g, 0.386 mmol) was converted to the hydrochloride salt by dissolving in water (2 mL) and the pH was adjusted to approximately 3.0 using 1 M HCl. Water was removed in vacuo and the residue was crystallized from aqueous EtOH to give compound 16-6 as a hydrochloride salt (71.0 mg). mp 243 [deg.] C (dec);

¹⁹ F NMR (DMSO-d ₄ ):? 1.69 (m). Analysis calculated for _{C 10 H 14 FN 3 O 4} .HCl: C, 40.62; H, 5.11; N, 14.21. Measured: C, 40.80; H, 5.09; N, 14.23.

Biological example

Black protocol

High throughput replicon assay (HTBS)

Replicon cells with H77 (genotype 1a) or Con1 (genotype 1b) HCV RNA and Renilla luciferase reporter cells were grown at a density of 1.6 x 10 &lt; 3 &gt; cells per well in 90 mL DMEM culture medium, excluding G- - well black plate. Compounds were diluted with serial in 100% DMSO and added to the cells with a 1: 225 dilution to achieve a final volume of 0.44% DMSO in a total volume of 90 μl using a Biotek μflow Workstation. Cell plates were incubated with 5% CO ₂ for 3 days at 37 ° C, after which the culture medium was removed and cells were assayed for luciferase activity as a marker for replication level. Expression of luminescent enzyme was measured using a Dual-Glo luminescent enzyme assay reagent (Promega, Madison, Wis.). Briefly, cells were lysed for 10 min by adding 20 μl of Dual-Glo luminescence enzyme buffer and then 20 μl of diluted Dual-Glo Stop & Glo substrate (1: 100) was added to each well. After incubation for 10 minutes, the luminescent signal was measured on a Perkin Elmer Envision Plate reader. The luminescence enzyme levels were converted to percentages for the untreated control (defined as 100%) and the data were analyzed using the exponential dose response equation y = a / (1+ (x / b)) using XLFit4 software (IDBS, Emeryville, CA) ) c). EC ₅₀ values were calculated from the equation.

Alternatively, the antiviral activity can be analyzed by HCV NS3 Protease IC ₅₀ Determination. HCV NS3 protease activity was determined by the Taliani method (Taliani M, Bianchi E, Narjes F, Fossatelli M, Urbania A, Steinkuhler C et al., A continuous assay or hepatitis C virus protease based on resonance energy transfer depsipeptide substrates. (FRET) depressed peptide substrate (RET S1, Anaspec, San Jose, Calif.), Based on a standard fluorescence resonance energy transfer (FRET) Lt; / RTI &gt;

Briefly, 2-10 nM of the purified NS3 protease domain was digested in 40% glycerol buffer with 50 mM HEPES pH 7.5 and 10 mM DTT using a 20 [mu] M isogenic NS4A peptide cofactor (Sigma, St. Louis, MO) Min at &lt; RTI ID = 0.0 &gt; 37 C. &lt; / RTI &gt; Compounds were diluted 1: 3 as a serial in DMSO, incubated for 10 min with an enzyme / cofactor mixture and the reaction was initiated by the addition of 2 [mu] M RET S1 substrate (final concentration). Fluorescence increments were continuously measured over 1 hour using a Victor 3 V fluorescent plate reader. The initial velocity was calculated for each inhibitor concentration using Workout 1.5 software (DAzDAQ, East Sussex, UK) using the maximum slope algorithm. Non-linear regression was performed to convert the rate data to a percentage of the untreated control (defined as 100%) and calculate the 50% inhibitory concentration (IC ₅₀ ).

NS3 Enzymatic Efficacy: The purified NS3 protease was complexed with the NS4A peptide and then incubated with a series of diluted compounds (using DMSO as the solvent). The reaction was initiated by addition of double-labeled peptide substrate and generation kinetics and the increase in fluorescence was measured. The IC ₅₀ was calculated by performing a non-linear regression of the velocity data. Activity against genotype 1b protease was initially tested. Depending on the efficacy obtained against genotype 1b, additional genotypes (1a, 2a, 3) and / or protease inhibitor resistant enzymes (D168Y, D168V, or A156T mutants) can be tested. BILN-2061 was used as a control for all assays. Compounds of the Examples were evaluated with this assay and found to have IC ₅₀ values of less than about 1 μM.

Replikon efficacy and cytotoxicity : Huh-luc cells (stably replicating I389 luc-ubi-neo / NS3-3 '/ ET genotype 1b replicon of Bartenschlager) were incubated for 72 hours with a series of diluting compounds (DMSO Lt; / RTI &gt; The amount of replicon was measured by bioluminescence and non-linear regression was performed to calculate the EC ₅₀ . A parallel plate treated with the same drug dilution was purchased from Promega CellTiter-Glo Cell viability assays were used to assess cytotoxicity. Depending on the efficacy achieved against 1b replicon, the compounds could be tested against genotype 1a replicons and / or inhibitor resistant replicons encoding D168Y or A156T mutations. BILN-2061 was used as a control for all assays. Compounds of the Examples were evaluated with this assay and found to have an EC ₅₀ value of less than about 5 μM.

Effect of Serum Protein on Replikin Efficacy

The replicon assay was performed in normal cell culture medium (DMEM + 10% FBS) supplemented with physiological concentrations of human serum albumin (40 mg / mL) or a-acid glycoprotein (1 mg / mL). It was compared with the EC ₅₀ in normal medium to determine the fold shift in the EC ₅₀ to the side effect in the presence of human serum proteins.

Enzyme Selectivity: Inhibition of mammalian proteases including 'Porcine Pancreatic Elastase', 'Human Leukocyte Elastase', 'Protease 3', and 'Cathepsin D' was measured at K _m for each substrate for each enzyme. The IC ₅₀ for each enzyme was compared to the IC ₅₀ obtained with the NS3 1b protease to calculate the selectivity.

MT-4 cell cytotoxicity: MT4 cells were treated with a series of diluent compounds for a period of 5 days. Cell viability was measured at the end of the treatment period using the Promega CellTiter-Glo assay and non-linear regression was performed to calculate CC ₅₀ .

The compound concentration in the cell associated with EC _50: Huh-luc the culture was incubated with the compound at the same concentration as EC _50. At various time points (0-72 hours), cells were washed 2X with cold media and extracted with 85% acetonitrile; Samples of media at each time point were also extracted. Cells and medium extracts were analyzed by LC / MS / MS to determine the molar concentration of the compounds in each fraction.

Solubility and Stability: An aliquot of the 10 mM DMSO stock solution was taken and the compound prepared in a test medium solution (PBS, pH 7.4 and 0.1 N HCl, pH 1.5) with a total DMSO concentration of 1% at a final concentration of 100 μM The solubility was determined. The test medium solution was shaken for 1 hour and incubated at room temperature. The solution was then centrifuged and the recovered supernatant was assayed on HPLC / UV. The solubility can be calculated by comparing the amount of compound detected in the test solution specified with the amount detected in DMSO at the same concentration. The solubility of the compounds after 1 hour incubation in test media at 37 占 폚 was also determined.

Stability in low-temperature preserved human, dog and rat hepatocytes:

Each compound was incubated at 37 占 폚 for 1 hour or less in hepatocyte suspension (100 占 퐇, 80,000 cells per well). Cold-preserved hepatocytes were reconstituted in serum-free incubation medium. The suspension was transferred to a 96-well plate (50 [mu] l / well). The compound was diluted in the incubation medium to 2 [mu] M incubation medium and then added to the hepatocyte suspension to start the incubation. Samples were taken after 0, 10, 30 and 60 minutes of incubation and the reaction quenched using a mixture of 0.3% formic acid in 90% acetonitrile / 10% water. The concentration of the compound in each sample was analyzed using LC / MS / MS. The half-life of disappearance of the compounds in the hepatocyte suspension was determined by fitting the concentration-time data using a single-exponential exponential equation. In addition, the data have gradually increased in scale to inherent liver clearance and / or total clearance.

Stability in Hepatic S9 Fraction in Human, Dog and Rat: Each compound was incubated at 37 째 C (n = 3) in S9 suspension (500,, 3 mg protein / mL) for less than 1 hour. The compound was added to the S9 suspension to start the incubation. Samples were taken at 0, 10, 30, and 60 minutes after initiation of incubation. The concentration of the compound in each sample was analyzed using LC / MS / MS. The half-life of disappearance of the compound in the S9 suspension was determined by fitting the concentration-time data using a single-stream exponential equation.

Cacao-2 transmission:

Both transmission directions (A to B) and reverse (B to A) transmissions were measured. A single layer of cacao-2 was grown to confluence on a collagen-coated, microporous, polycarbonate membrane in a 12-well Costar Transwell® plate. The compound was administered to the side of the apex for the directional permeability (from A to B) and was administered on the basolateral membrane for the reverse permeability (from B to A). Using a 5% CO ₂ the cells were incubated in a humid incubator at 37 ℃. At the beginning of the incubation, 1 hour and 2 hours after the incubation, 200-μl aliquots were taken from the receiving chamber and replaced with fresh assay buffer. The concentration of the compound in each sample was determined using LC / MS / MS. The apparent permeability (Papp) was calculated.

Plasma protein binding: Plasma protein binding was measured by equilibrium dialysis. Each compound was spiked into blank plasma at a final concentration of 2 [mu] M. The spiked plasma and phosphate buffer was placed on the opposite side of the assembled dialysis cells and then slowly rotated in a 37 ° C water bath. At the end of the incubation, the concentration of the compound in plasma and phosphate buffer was determined. The percent unbinding was calculated by the equation:

[Wherein,

C _f and C _b is the free concentration and binding concentration, respectively, as measured as buffer and plasma concentrate after dialysis.

CYP450 profiling: Each compound was incubated with five recombinant human CYP450s, including CYP1A2, CYP2C9, CYP3A4, CYP2D6 and CYP2C19, in the presence and absence of NADPH. A series of samples could be taken from the incubation mixture at the start of incubation, 5, 15, 30, 45 and 60 minutes after the start of incubation. The concentration of the compound in the incubation mixture was measured by LC / MS / MS. Percentage of compound remaining after incubation at each time point was calculated by comparison with sampling at the start of incubation.

Stability in rats, dogs, monkeys and human plasma: Compounds were incubated in plasma (rats, dogs, monkeys or humans) for 2 hours or less at 37 ° C. The compounds were added to plasma at a final concentration of 1 and 10 mg / mL. The aliquots were collected at 0, 5, 15, 30, 60, and 120 minutes after compound addition. The concentrations of the compounds and major metabolites were measured by LC / MS / MS at each time point. Biological data (antiviral efficacy [EC ₅₀ ] determined using Renilla luciferase (RLuc) -compliant HCV replicon reporter assay - HCV 1b RLuc).

Biological Example 1 : Anti-HCV activity of the combination of Compound 1 and Compound 2

Materials and methods

Compound 1 and Compound 2 were synthesized in Gilead Sciences, Foster City, CA.

Cell line

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany). The replicons in such cells are determined by I389luc-ubi-neo / NS3-3 '/ ET and encode a firefly luciferase reporter gene as well as a selectable resistance marker (neomycin phosphotransferase). Huh-luc cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO, Carlsbad, Calif.) Supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and 0.5 mg / mL G-418 . Cells were subcultured twice a week and maintained at the subconfluent level.

EC ₅₀ determination

The replicon cells were seeded into 96 well plates at a density of 5 x 10 ³ cells per well in 100 μl of DMEM culture medium except for G418. Compounds 1 and 2 were serially diluted 1: 3 in 100% DMSO (Sigma). Such a chain dilution was added to the cells at a 1: 200 dilution to achieve a final concentration of 0.5% DMSO in a final volume of 200 [mu] l. The plates were incubated at 37 ° C for 3 days, then the culture medium was removed, the cells were dissociated and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, WI). The level of HCV replication in the drug treated samples was expressed as a percentage of that of the untreated control (defined as 100%) and was calculated using the algebraic dose response equation y = a / (1+) using XLFit4 software (IDBS, Emeryville, Calif. (x / b) c). EC ₅₀ values were calculated from the resulting equations as described in the prior art (Delaney, WE, et al., Antimicrobial Agents Chemotherapy, 45 (6): 1705-1713 (2001)).

Antiviral combination study

The replicon cells were seeded in a 96-well plate at a density of 5 x 10 ³ cells per well in 100 μl of the culture medium. Compounds 1 and 2 were sequentially diluted in 100% DMSO as described above and added to the 96-well plate in matrix format to provide a defined set of drug concentrations at a final volume of 200 μl and a final DMSO concentration of 0.5% do. For each individual drug, the EC ₅₀ value was chosen as the median for the concentration range tested. Cells were incubated for 3 days and analyzed for luciferase expression as described above. For combination studies, two independent trials of three were performed.

Combination data analysis

Data were analyzed using a MacSynergy II program developed by Prichard and Shipman (Prichard MN, Aseltine KR, Shipman C, Jr., MacSynergy ^TM II, Version 1.0, University of Michigan, Ann Arbor, Michigan, Prichard MN, Shipman C, Jr., Antivir Ther 1 (1): 9-20 (1996); Prichard MN, et al. The software calculates the theoretical inhibition values assuming additive interactions between drugs (based on the Bliss Independence model), and theoretically and observed inhibition Quantifying the statistically significant difference between the values. By plotting such differences in three dimensions, convexities in the Z-plane exhibit antiviral synergism, while concavities show surface antiviral antagonism between compounds The calculated volume of surface deviation is expressed as nM ² %. According to Prichard and Shipman, the combination effect is defined as:

부피 > 100 nM² 인 경우, 매우 상승작용적임.

Very high if volume> 100 nM ² .

부피가 > 50 및 ≤100 nM² 인 경우, 약간 상승작용적임.

Slightly synergistic if the volume is> 50 and ≤ 100 nM ² .

부피가 > -50 nM² 및 ≤50 nM² 인 경우, 가산성임.

Additive if volume is> -50 nM ² and ≤ 50 nM ² .

부피가 > -100 nM² 및 ≤-50 nM² 인 경우, 약간 길항작용적임.

Volume > -100 nM &lt; ² &^gt; When ≤-50 nM ² , it is slightly antagonistic.

부피가 ≤-100 nM² 인 경우, 길항작용적임.

When the volume is ≤-100 nM ² , it is antagonistic.

result

Before starting the combination experiment, EC ₅₀ values in Huh-luc replicon cells were determined for Compound 1 and Compound 2, and the results are shown in Table II. Both compounds had antiviral effects.

Table II

Individual EC ₅₀ for anti-HCV Compounds 1 and 2 in Huh-luc replicon cells

^a EC ₅₀ represents the mean ± SD for two or more independent experiments.

The antiviral effect of the combination of Compound 1 and Compound 2 was measured and the resulting data was analyzed using MacSynergy II to provide a surface graph showing significant deviations from additivity. The quantification of statistically significant horses from additions showed that the combination of compounds 1 and 2 had a synergistic / antagonistic volume of -50 nM ² to 50 nM ² , and as shown in Table III, Effect.

Table III

Antiviral synergistic and antagonistic action on the combination of Compound 1 and Compound 2 and quantification of drug interaction

^a values represent the mean ± standard deviation of two independent experiments in triplicate

The results of the in vitro experiments presented in Table III show that Compound 2 has additive antiviral activity when combined with Compound 1.

Biological Example 2 : Combination with Compound 3

Materials and methods

Antiviral compound

Compound 1 and Compound 3 were synthesized in Gilead Sciences, Foster City, CA. Ribavirin and IFN-a were purchased from Sigma (St. Louis, MO).

Cell line

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany). The replicons in such cells are designated I389luc-ubi-neo / NS3-3 '/ ET and encode the firefly luciferase reporter gene as well as the selectable resistance marker (neomycin phosphotransferase) . The Huh-luc cells 10% fetal bovine serum (FBS, Hyclone, Logan, UT ) and GlutaMAX supplemented with G-418 (Invitrogen) in ^{0.5 mg / mL TM (Invitrogen,} Carlsbad, CA) is Dulbecco's Modified Eagle Medium with ( D-MEM). Cells were subcultured twice a week and maintained at the subconfluent level.

EC ₅₀ determination

The replicon cells were seeded in 96-well plates at a density of 5 x 10 ³ cells per well in 100 μl of DMEM and 10% FBS culture medium, except for G-418. Compounds were serially diluted 1: 3 in 100% DMSO (Sigma). Such chain dilutions were added to the cells at a 1: 200 dilution to a final concentration of 0.5% DMSO in a total volume of 200 μl. The plates were incubated at 37 ° C for 3 days, then the culture medium was removed, the cells were dissociated and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, WI). HCV replication levels in drug-treated samples were expressed as percent of their untreated control (defined as 100%) and data were analyzed using the logistic dose response equation y = a / (1) using XLFit4 software (IDBS, Emeryville, Calif. + (x / b) ^c ). EC ₅₀ values were calculated from the equations presented as the results described above.

Antiviral combination study

The replicon cells were seeded in 96-well plates at a density of 5 x 10 ³ cells per well in 100 μl of culture medium, except for G-418. Compound 3 and other compounds were successively diluted in 100% DMSO as described above and added to the 96-well plate in matrix format to give a defined set of drug concentrations and ratios that differed between 200 μl final volume and 0.5% final DMSO concentration . For each individual drug (except ribavirin), the EC ₅₀ value was chosen as the median for the concentration range tested. For ribavirin, the selected dose of 6.2 μM was selected because 6.2 μM was less than about three times the concentration at which cytotoxicity was observed. Cells were incubated with drug for 3 days and analyzed for luciferase expression as described above. For each combination study, two independent experiments were performed in triplicate.

Combination data analysis

Data were analyzed using the MacSynergy II program developed by Prichard and Shipman. The software assumes the additive interactions between drugs (based on the Bliss Independence model) to yield the theoretical inhibition and quantifies the statistically significant difference between the theoretical and experimental inhibition values. By plotting such differences in three dimensions, convexities in the Z-plane exhibit antiviral synergism, while concaves provide a surface showing antiviral antagonism between the compounds. The calculated volume of the surface deviation is expressed as nM ² %. According to Prichard and Shipman, the combination effect is defined as:

부피 > 100 nM² 인 경우, 강력한 상승작용; 이러한 분량의 상승작용은 아마도 생체내에서 중요할 수 있음

Strong synergism when volume> 100 nM ² ; This amount of synergy may be important in vivo

부피가 > 50 및 ≤100 nM² 인 경우, 중도의 상승작용; 이러한 분량의 상승작용은 생체내에서 중요할 수 있음

Moderate synergism when the volume is > 50 and &lt; 100 nM ² ; This amount of synergy can be important in vivo

부피가 > 25 nM² 및 ＜50 nM² 인 경우, 경미한 상승작용.

Mild synergism when volume is> 25 nM ² and <50 nM ² .

부피가 > -25 nM² 및 ≤25 nM² 인 경우, 부가적임

Additional if volume is> -25 nM ² and <25 nM ²

부피가 > -25 nM² 및 > -50 nM² 인 경우,

If the volume is> -25 nM ² and> -50 nM ^2,

부피가 > -100 nM² 및 ≤-50 nM² 인 경우, 중도의 길항작용; 이러한 분량의 길항작용은 생체내에서 중요할 수 있음.

Volume > -100 nM &lt; ² &^gt; ≤-50 nM ² , moderate antagonism; This amount of antagonism may be important in vivo.

부피가 ≤-100 nM² 인 경우, 강력한 길항작용; 이러한 분량의 길항작용은 아마도 생체내에서 중요할 수 있음.

When the volume is ≤-100 nM ² , strong antagonism; This amount of antagonism may be important in vivo.

result

EC ₅₀ values for individual compounds in Huh-luc replicon cells.

Prior to the start of the combination experiment, the EC ₅₀ value in Huh-luc replicon cells was determined as shown in Table IV. All compounds except ribavirin, which did not have antiviral activity, had antiviral activity below the concentration at which they started showing cytotoxicity.

Table IV

Individual EC ₅₀ for anti-HCV compounds in Huh-luc replicon cells

^a EC ₅₀ represents the mean ± SD for two or more independent experiments.

^b INF-α EC ₅₀ is expressed in units per milliliter (mL) instead of nanolomole concentration (U).

Combination antiviral effects and drug interactions

The anti-viral effects of IFN-a, ribavirin, and Compound 3 when combined with Compound 1 were assayed. The resulting data was analyzed using MacSynergy II, which provides a surface graph showing significant deviations from additivity. The quantification of statistically significant deviations from additive showed that the combination of compound 3 and IFN-? Provided a slight synergism (synergistic volumes of 32 and 36.5 nM ² , respectively, Table V). The combination of compound 3 with the non-nucleoside NS5B inhibitor compound 1 provided a synergistic volume of 14.5 nM ² indicating additive antiviral interaction. As shown in Table V, none of the compounds provided an antiviral antagonist volume outside the additive range (> -25 nM &lt; ² &gt;

Table V

Antiviral synergy and antagonism of drug combinations with compound 3 and quantification of drug interactions

^a values represent the mean ± standard deviation of independent experiments performed twice in triplicate

Such in vitro antiviral combination experiments indicate that the novel HCV NS3 protease inhibitor compound 3 has a slight synergy when combined with IFN- [alpha] and a moderate synergistic effect when combined with ribavirin. The results show that Compound 3 can potentially be used in combination with current standards of care in HCV patients (PEG-IFN-a and ribavirin) to achieve enhanced viral load inhibition without diminishing the efficacy of any individual drug . The combination of the non-nucleoside of Compound 3 (Compound 1) with the NS5B polymerase inhibitor resulted in additivity. Such results indicate that Compound 3 may be suitable for drug combination probing comprising multiple classes of specific HCV inhibitory factors in a patient.

Biological Example 3: Compound 4 combination

Materials and methods

Anti-HCV agonists

Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 6 were synthesized in Gilead Sciences (Foster City, Calif.). Puromycin, IFN-a and ribavirin were purchased from Sigma (St. Louis, Mo.). Calcein AM was purchased from Anaspec (Fremont, CA).

Cell line and cell culture

The HCV genotype 1a replicon cell line used in this study has been described above. Cells were resuspended in 10% FBS (HyClone, Logan, UT, Cat # SH30071.03), 100 Units / mL penicillin, 100 ug / mL streptomycin (Gibco, Carlsbad, CA, Cat # 15140-122) Were grown in cell culture media containing Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX (Gibco, Carlsbad, CA, Cat # 10569-044) supplemented with amino acids (Gibco, Carlsbad, CA, Cat # 11140-050) . Replicon cells were maintained in 0.5 mg / mL Geneticin (Invitrogen, Carlsbad, CA, Cat # 10131-035) to prevent loss of HCV replicon. Cells were subcultured every 3-4 days before reaching a confluent state.

EC ₅₀ , CC ₅₀ HCV replicon assay for determination and combination studies

All compounds were fed into 100% DMSO except for IFN- [alpha] fed into the buffer specified in the manufacturer (Sigma, St. Louis, MO, Cat # I4276). Compound chain dilutions were performed in 100% DMSO except for IFN-a which was cascade diluted in the cell culture medium described in section 3.2. All chain dilutions were performed on 384-well polypropylene plates (Thermo Scientific, Hudson, NH, Cat # 4341) using Biomek FX Workstations. For EC ₅₀ and CC ₅₀ determinations, the test compound was serially diluted in 10 steps in a 1: 3 dilution in columns 3 to 20 of a 384-well plate. For the study of the combination, one compound was serially diluted in nine steps in a 1: 2 dilution in the horizontal direction while the other compound was serially diluted in seven steps in a 1: 2 dilution in the vertical direction . This provided a fixed set of different drug concentrations and ratios. For each individual drug, the EC ₅₀ value was chosen to be the median of the concentration range tested. All chain dilutions were performed in duplicate in the same 384-well plate per compound. 100% DMSO was added to columns 1 to 2 of each chain dilution 384-well plate. 100 [mu] M of HCV protease inhibitor ITMN-191 was added to column 34 as a 100% inhibitory control of HCV replication while 10 mM of puromycin was added to column 24 as a 100% cytotoxic control.

In each well of a black polystyrene 384-well plate (Greiner Bio-one, Monroe, NC, Cat # 781086, treated cell culture), 90 [mu] l cell culture medium containing 2000 suspended HCV replicon cells Geneticin absence) was added using a Biotek μFlow Workstation. For compound transport to the cell culture plates, 0.4 μl of the compound solution from the compound chain dilution plate was transferred to a cell culture plate on a Biomek FX Workstation. The concentration of DMSO in the final assay well was 0.44%. The plates were incubated at 37 ℃ 3 days under 5% CO ₂ and 85% humidity.

The HCV replicon assay is a multiple complex assay that can assess both cytotoxicity and anti-replicon activity from the same well. The CC ₅₀ test was performed first. The medium in the 384-well cell culture plate was aspirated and the wells were washed 4 times with 100 [mu] l 1X PBS each using a Biotek ELX405 plate washer. 50 μl of a solution containing 400 nM calcein AM (Anaspec, Fremont, CA, Cat # 25200-056) in 1 X PBS was added to each well of the plate using a Biotek μFlow Workstation. The plate was incubated for 30 minutes at room temperature and the fluorescence signal (excitation 490 nm, emission 520 nm) was measured using a Perkin Elmer Envision Plate Reader.

EC ₅₀ assays were performed in the same well as CC ₅₀ assays. The calcein-PBS solution in 384-well cell culture plates was aspirated using a Biotek ELX405 plate washer. A volume of 20 [mu] l of Dual-Glo Luciferase Buffer (Promega, Madison, Wis., Cat # E298B) was added to each well of the plate using a Biotek 占 lowlow Workstation. The plate was incubated at room temperature for 10 minutes. Then a volume of 20 μl containing a 1: 100 mixture of Dual-Glo Stop & Glo substrate (Promega, Madison, WI, Cat # E313B) and Dual-Glo Stop & Glo Buffer (Promega, Madison, WI, Cat # E314B) Was added to each well of the plate using a Biotek 占 Flow Workstation. Subsequently, the plate was incubated at room temperature for 10 minutes, and the luminescence signal was measured with a Perkin Elmer Envision Plate Reader.

Data Analysis

Cytotoxic effects were measured by calcein AM conversion to fluorescent products. Percent cytotoxicity was calculated as Equation 1:

Wherein X _C is the fluorescence signal from the compound-treatment well; M _B is the mean fluorescence signal from the puromycin-treatment well; M _D is the mean fluorescence signal from the DMSO-treated well. Anti-HCV replication activity was determined by the luminescent signal generated from the reporter lane luciferase of HCV replicon. The percent inhibition for HCV replicon was calculated using Equation 1, where X _C is the emission signal from the compound-treatment well; M _B is the average emission signal from the ITMN-191-treatment well; M _D is the mean emission signal from the DMSO-treatment well.

The CC ₅₀ value was determined as the test compound concentration resulting in a 50% reduction in cell viability. The EC ₅₀ value was determined as the test compound concentration resulting in a 50% reduction in HCV replication. The CC ₅₀ and EC ₅₀ values were all obtained by nonlinear regression fitting of the experimental data for equation 2 using the Pipeline Pilot 5.0 software package (Accelrys, San Diego, Calif.):

Wherein y is the observed% inhibition of HCV replicon at concentration x of the compound; d is the response estimated at compound concentration 0; a is the response estimated at an infinite compound concentration; c is the mid-range concentration (CC ₅₀ or EC ₅₀ ); b is the Hill slope factor.

Combination study experiment data were analyzed using the MacSynergy II program developed by Prichard and Shipman. The software (MacSynergy II, University of Michigan, MI) calculates the theoretical inhibition assuming additive interactions between drugs (based on Bliss Independence model), and statistically significant Quantify the difference. By plotting such differences in three dimensions, convexities in the Z-plane exhibit antiviral synergism, while concaves provide a surface showing antiviral antagonism between the compounds. The calculated volume of the surface deviation is expressed as nM ² %. According to Prichard and Shipman, the combination effect is defined as:

강력한 상승작용: > 100 nM²%

Strong synergism:> 100 nM ² %

중도의 상승작용: > 50 및 ≤100 nM²%

Moderate synergy:> 50 and ≤100 nM ² %

경미한 상승작용: > 25 및 ≤ 50 nM²%

Mild synergism:> 25 and ≤ 50 nM ² %

가산성: ≤ 25 및 > -25 nM²%

Additive: ≤ 25 and> -25 nM ² %

경미한 길항작용: ≤ -25 및 > -50 nM²%

Mild antagonism: ≤ -25 and> -50 nM ² %

중도의 길항작용: ≤ -50 및 >-100 nM²%

Moderate antagonism: ≤-50 and> -100 nM ² %

강력한 길항작용: ≤ -100 nM²%

Strong antagonism: ≤ -100 nM ² %

For each combination study, three independent experiments were performed, four each for each experiment.

result

Antiviral activity and cytotoxicity of individual compounds in the HCV genotype 1a replicon assay.

The anti-HCV activity and cytotoxicity of Compound 4 and other compounds were tested in Huh-7 cells bearing the HCV genotype 1a replicon. EC ₅₀ and CC ₅₀ values are listed in Table VI. No significant cytotoxicity was observed for all compounds up to the highest concentration tested.

TABLE VI

The EC ₅₀ and CC ₅₀ of the compounds used in this study on HCV genotype 1a replicons

^a values are the mean ± standard deviation for three or more independent experiments.

^{b The} IFN-α value is expressed in units per milliliter (mL) instead of the nanomolar concentration (U).

Antiviral activity and cytotoxicity in combination with other classes of compounds 4 with anti-HCV agonists.

The antiviral effect of compound 4 in combination with other anti-HCV compounds was evaluated using HCV genotype 1a replicons. The results were analyzed using MacSynergy II, which provides a surface graph showing significant deviations from additivity. The synergistic and antagonistic volumes (nM ² %) calculated from deviations from the additive surface are summarized in Table VII. When compound 4 is combined with IFN-a, compound 2 and compound 6 in a 95% confidence interval, the average synergistic and antagonistic volumes indicating the additive interaction with such compounds are 25 to -25 nM &lt; ² &gt; Respectively. Furthermore, Compound 4 exhibited a synergistic volume in the range of 25 to 50 nM ² % when combined with Compound 1, Compound 5 or Compound 3, suggesting a slight synergistic interaction.

TABLE VII

Antiviral synergy and antagonism of drug combinations with compound 4 and quantification of drug interactions

Values represent the mean ± standard deviation of independent experiments performed in triplicate

As shown in Table VIII, cell viability in all combination studies exceeded 85% at all concentration ratios, and all drug combinations showed additive effects on cytotoxicity.

Table VIII

Cytotoxic synergy and antagonism of drug combinations with compound 4 and quantification of drug interactions

^a values represent the mean ± standard deviation of independent experiments performed in triplicate

However, Compound 4 exhibited an antagonistic activity volume of -43 nM ² % in combination with ribavirin, suggesting a slight antagonistic interaction.

Table IX

Cytotoxic synergy and antagonism of drug combinations with ribavirin and quantification of drug interactions

^a values represent the mean ± standard deviation of independent experiments performed in triplicate

The concentration of ribavirin exhibiting the highest antagonistic activity with compound 4 is about 0.5 to 1 [mu] M, which is about 10-fold greater than the steady-state plasma concentration of ribavirin (6-11 [mu] M) observed in humans at a dose of 800 mg / low. At such physiological concentrations of ribavirin (6-11 [mu] M), the antagonistic interaction between ribavirin and compound 4 crosses at a minimum in a broad range of compound 4 concentrations (0 to 0.44 [mu] M). Thus, the mild antagonism observed between ribavirin and compound 4 in the in vitro replicon system is less likely to have clinical significance.

conclusion

(IFN-a / ribavirin) as well as internal development candidate Drug Compound 1 and Compound 5 (non-nucleoside NS5B inhibitor) of Gilead Sciences, as well as the current standard of care for the antiviral activity of Compound 4 (enantiomeric mixture) 2 and Compound 3 (NS3 protease inhibitor), and Compound 6 (NS5A inhibitor). As summarized in Table VIII, Compound 4 exhibited additive antiviral activity in combination with IFN- [alpha] / Compound 2 and Compound 6, and showed slight synergism with Compound 1, Compound 5 and Compound 3. [

The combination of compound 4 with ribavirin provided mild antagonism at a ribavirin concentration of 0.5 to 1 [mu] M, which is about 10-fold lower than its normal-state physiological concentration (6-11 [mu] M) in human plasma. At clinically relevant ribavirin concentrations, the antagonistic interactions between the two compounds became negligible.

Biological Example 4 : Compound 5 combination

The antiviral activity of Compound 5 was determined by using the internally developed Compound Compound 1, Compound 2, and Compound 3 (NS3 protease inhibitor), GT-1b Huh-lunet cells (using substantially the same method as that for Compound 4) 6 (NS5A inhibitor), Compound 4 (C-nuc NS5B inhibitor) and also with the approved HCV therapeutic agents PEG-IFN-a and ribavirin. Combination data was analyzed using MacSynergy II software based on the Bliss Independence model. (NM ² ), calculated from 95% confidence levels, from two independent experiments with three results of the combination test. The combination effect is defined as:

부피 > 100 nM² 인 경우 강력한 상승작용; 이러한 분량의 상승작용은 아마도 생체내에서 중요함.

Strong synergism at volume> 100 nM ² ; This amount of synergy is probably important in vivo.

부피 > 50 이고 ≤ 100 nM² 인 경우 중도의 상승작용; 이러한 분량의 상승작용은 생체내에서 중요할 수 있음.

Moderate synergism when volume> 50 and ≤ 100 nM ² ; This amount of synergy can be important in vivo.

부피 > 25 이고 < 50 nM² 인 경우 경미한 상승작용

Minimal synergism with volume> 25 and <50 nM ²

부피 > -25 이고 ≤ 25 nM² 인 경우 부가성

Additive in case of volume> -25 and ≤ 25 nM ²

부피 < -25 이고 > -50 nM² 인 경우 경미한 길항작용

Mild antagonism with volume <-25 and> -50 nM ²

부피 > -100 nM² 이고 ≤ -50 nM² 인 경우 중도의 길항작용; 이러한 분량의 길항작용은 생체내에서 중요할 수 있음.

Moderate antagonism when volume> -100 nM ² and ≤-50 nM ² ; This amount of antagonism may be important in vivo.

부피 ≤ -100 nM² 인 경우 강력한 길항작용; 이러한 분량의 길항작용은 아마도 생체내에서 중요함.

Strong antagonism when volume ≤ -100 nM ² ; This amount of antagonism is probably important in vivo.

The combination of allosteric NS5B inhibitor compound 1 and compound 5 provides a moderate synergism in the replicon assay. Studies with other HCV inhibitors, including PEG-IFN-a and ribavirin, have demonstrated additive effects on mild synergistic interactions.

TABLE X

1b Antiviral effect of Compound 5 in combination with other anti-HCV drugs in Huh-luc replicon cells

^a. Values represent the mean ± standard deviation of independent experiments performed twice in triplicate

Biological Example 5 : Compound 6, combination

Materials and methods

compound

Compound 1, Compound 2, Compound 3, Compound 6 and Compound 7 were synthesized in Gilead Sciences (Foster City, CA). IFN- [alpha] and ribavirin were purchased from Sigma (St. Louis, MO).

Cell line

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany). The replicons of these cells were designated I389luc-ubi-neo / NS3-3 '/ ET and encode the firefly luciferase reporter gene as well as the selectable resistance marker (neomycin phosphotransferase). Huh-luc cells were treated with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1X penicillin / streptomycin, 1X nonessential amino acids and 0.5 mg / ml G-418 (all from Invitrogen, Carlsbad, CA) (Dulbecco ' s Modified Eagle ' s Medium GlutaMax (DMEM; Invitrogen, Carlsbad, Calif.). Cells were subcultured twice weekly and maintained at the subconfluent level.

black

Antiviral activity assay in HCV Huh-luc replicon cells

The replicon cells were seeded into 96-well plates at a density of 7 x 10 ³ cells per well in 100 μl of DMEM culture medium except for G-418. The compounds were serially diluted 1: 2 in 100% DMSO. Serial dilutions were added to the cells at a 1: 200 dilution to achieve a final concentration of 0.5% DMSO in a total volume of 200 μl. The plates were incubated at 37 ° C for 3 days, then the culture medium was removed, the cells were dissociated and assayed for luciferase activity using a commercial luciferase assay (Promega, Madison, WI).

Antiviral combination study

The replicon cells were seeded into 96-well plates at a density of 7 x 10 ³ cells per well in 100 μl culture medium except for G-418. Compound 6 and other compounds were sequentially diluted 1: 2 in 100% DMSO and added in a matrix format to a 96-well plate to obtain a defined set of drug concentrations and ratios at a final volume of 200 μl and a final DMSO concentration of 0.5% . For each individual drug, the EC ₅₀ value was chosen to be the median of the concentration range tested. Cells were incubated for 3 days and assayed for luciferase expression using commercial luciferase assay (Promega). For each combination study, two independent trials of three were performed.

Cell cytotoxicity determination

The replicon cells were seeded and treated with the drug as described for the antiviral combination studies. After 3 days of incubation at 37 ° C, the culture medium was removed, the cells were dissociated and assayed for cytotoxicity using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Relative Light Units were converted to percentages for the untreated control (defined as 100%).

Data Analysis

EC ₅₀ yield

Following EC ₅₀ assays, luciferase levels in drug-treated samples were expressed as their percentages in the untreated control (defined as 100%). EC ₅₀ values were calculated by nonlinear regression analysis of repeated data sets using XLfit 4 software (IDBS, Emeryville, Calif.).

Production of antiviral synergism and antagonism

Following the combination assay, luciferase levels in the drug-treated samples were expressed as their percentages in the untreated control (defined as 100%). The repeated data set was then analyzed using the MacSynergy II program developed by Prichard and Shipman. The software assumes the additive interactions between drugs (based on the Bliss Independence model) to yield the theoretical inhibition and quantifies the statistically significant difference between the theoretical and experimental inhibition values. By plotting such differences in three dimensions, convexities in the Z-plane exhibit antiviral synergism, while concaves provide a surface showing antiviral antagonism between the compounds. The calculated volume of the surface deviation is expressed as nM ² %. According to Prichard and Shipman, the combination effect is defined as:

부피 >　100 nM² 인 경우, 강력한 상승작용; 이러한 분량의 상승작용은 아마도 생체내에서 중요함

Strong synergism when volume> 100 nM ² ; This amount of synergy is probably important in vivo

부피 >　50 및 ≤100 nM² 인 경우 중도의 상승작용; 이러한 분량의 상승작용은 생체내에서 중요할 수 있음

Moderate synergism in volume> 50 and ≤100 nM ² ; This amount of synergy can be important in vivo

부피 >　25 및 <　50 nM² 인 경우, 경미한 상승작용

In the case of volume > 25 and < 50 nM ² ,

부피 >　-25 nM2 및 ≤25 nM² 인 경우, 부가성

If the volume of> -25 nM2 and ≤25 nM ^2, additionality

부피 <　-25 및 >　-50 nM² 인 경우, 경미한 길항작용

For volumes < -25 and > -50 nM &lt; ² &gt;, slight antagonism

부피 >　-100 nM² 및 ≤-50 nM² 인 경우, 중도의 길항작용; 이러한 분량의 길항작용은 생체내에서 중요할 수 있음

Moderate antagonism when volume > -100 nM ² and &lt; - 50 nM ² ; This amount of antagonism may be important in vivo

부피 ≤ -100 nM² 인 경우, 강력한 길항작용; 이러한 분량의 길항작용은 아마도 생체내에서 중요함.

Strong antagonism when volume ≤ -100 nM ² ; This amount of antagonism is probably important in vivo .

result

Antiviral activity of individual compounds in Huh-luc replicon cells.

Prior to initiation of the combination experiment, the antiviral activity of the individual compounds was determined in Huh-luc replicon cells. EC ₅₀ values consistent with previous results were observed for all seven compounds.

Table XI

Individual EC ₅₀ values for anti-HCV compounds in Huh-luc replicon cells

^a EC ₅₀ represents the arithmetic mean ± standard deviation for three or more independent experiments.

^b IFN-α EC ₅₀ is expressed in units per milliliter (mL) instead of nanolomole concentration (U).

Combination antiviral effects and drug interactions

The antiviral effect of Compound 6 in combination with other HCV inhibitory factors was assessed using the HCV 1b replicon system. The resulting data was analyzed using MacSynergy II, which provides a surface plot showing significant variability of additivity. The quantification of statistically significant deviations from additivity from two independent experiments is summarized in Table XII. The combination of Compound 6 with IFN- [alpha] or Compound 1 gave a synergistic volume of 32 and 34 nM &lt; ² &gt;, respectively, indicating slight synergism. Ribavirin, Compound 2 and Compound 7 exhibited synergistic volumes of 61, 52 and 51, respectively, in combination with Compound 6, indicating a moderate synergistic interaction between Compound 6 and these three HCV inhibitory factors. The combination of compound 6 with compound 3 showed a synergistic volume of 132 nM ² %, indicating strongly synergistic antiviral interaction. None of the compounds in combination with Compound 6 provided an antiviral antagonist volume outside the additive range (&gt; -25 nM).

Table XII

Antiviral synergy and antagonism to drug combinations with Compound 6 and quantification of drug interactions

^a values represent the arithmetic mean ± standard deviation of independent experiments performed twice in triplicate.

The percentage of cell viability in combination with other HCV inhibitors of Compound 6

To ensure that the antiviral combination results were not confounded by the combination cytotoxicity, cytotoxicity was examined in parallel using the same compound concentrations tested in the antiviral assays (Table XIII). For all compounds, cell viability was> 98% of the untreated control for the combination at the highest concentration tested. Thus, no significant in vitro cytotoxicity was observed when compound 6 was tested alone or in combination with such agents.

Table XIII

Percent cell viability for Compound 6 combined in Huh-luc replicon cells

^a % cell viability represents the arithmetic mean ± standard deviation for independent experiments performed two or more times in triplicate.

^b IFN-α is expressed in units per milliliter (mL) instead of nano-molar concentration (U).

conclusion

These in vitro experimental results indicate that Compound 6 has a mild antiviral synergistic action when combined with IFN- [alpha] or non-nucleoside NS5B polymerase inhibitor compound 1. [ The combination of compound 6 with ribavirin, NS3 protease inhibitor compound 2 or nucleoside NS5B polymerase inhibitor compound 7 provided the result of acting a moderate antiviral synergistic effect. A strong antiviral synergistic effect was observed between the compound 6 and the NS3 protease inhibitor compound 3. No significant in vitro cytotoxicity was noted during testing of these drug combinations. These results suggest that Compound 6 can be reasonably combined with current care standards.

Biological Example 6:

compound

Compound 1, Compound 3, Compound 4, and Compound 6 were purchased from Gilead Sciences, Inc. of Foster City, CA. .

Cell line

HCV genotype 1b replicon cells (Huh-luc) were obtained from Reblikon (Mainz, Germany). The replicons of such cells are designated I389luc-ubi-neo / NS3-3 '/ ET and encode the firefly luciferase reporter gene as well as the selectable resistance marker (neomycin phosphotransferase). Huh-luc cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1X penicillin / streptomycin, 1X nonessential amino acids and 0.5 mg / ml G-418 (all from Invitrogen, Carlsbad, CA) (Dulbecco ' s Modified Eagle ' s Medium GlutaMax (DMEM; Invitrogen, Carlsbad, Calif.). Cells were subcultured twice weekly and maintained at the subconfluent level.

black

Determination of the compound concentration required to inhibit replicon RNA at 1 to 1.5 log during 6 days of treatment

Genotype 1b replicon cells were excluded from G418, ⁵  The cell / flask was seeded in T-75 flasks at the cell density. Compounds were individually added to the cells at various concentrations: Compound 6 was added at a concentration of 1 pM, 2 pM, 4 pM, 6 pM, 8 pM, or 12 pM and Compound 4 was added at a concentration of 125 nM, 250 nM, 375 nM, 500 nM or 1000 nM , Compound 1 was added at a concentration of 1.25 nM, 2.5 nM, 5 nM, 2.75 nM or 10 nM and Compound 3 was added at a concentration of 3.75 nM, 7.5 nM, 11.25 nM, 15 nM, 30 nM or 60 nM. The flask was incubated at 37 ° C, and the medium and compound were replaced every two days. After 6 days of incubation, replicon cells were harvested for RNA extraction and replicon RNA QRT-PCR analysis.

Compound combination replicon care black

Genotype 1b replicon cells were plated in a T-75 flask at 2.5 x 10 &lt; ⁵  Seeded at the density of the cells / flask. The compound was added to a T-75 flask at the following concentrations: 4 pM for Compound 6, 1000 nM for Compound 4, 10 nM for Compound 1, and 26.25 nM for Compound 3. The flask was incubated at 37 占 폚, and the compound and the medium were grounded every other day. All experiments were carried out in duplicate and indicated as Flask 1 and Flask 2. On day 6, all cells from Flask 1 were harvested for RNA extraction and HCV replicon-specific QRT-PCR analysis, and the cells from Flask 2 were replated on a 10 cm tissue culture dish for 14 days in the presence of G418 Colony formation of uncleaned replicon cells was recorded.

QRT-PCR assay

all  RNA was purified using a RiboPure kit (AM1924 Life Technologies Corporation  Carlsbad, CA) according to the manufacturer's protocol. The extracted RNA samples were stored at -80 ° C until use. For quantitative RT-PCR assays, a Qiagen One-step QRT-PCR kit was used according to the protocol of the manufacturer (Qiagen, Valencia CA). (SEQ ID NO: 1) and reverse NS3_180 5'-GGTCCTGGTCCACATTGGTGT-3 '(SEQ ID NO: 2) and 18S (SEQ ID NO: 2) as genotypic 1b HCV NS3 gene specific primers NS3_180FL 5'-CGGCGGACTGTCTATCATGGTGC [FAM] G- rRNA LUX ^TM  [FAM] The internal control primer set (115HM-01) was provided by Invitrogen corporation (Carlsbad, CA). For the reverse transcription step, the reaction was incubated at 44 DEG C for 30 minutes, and the reverse transcriptase enzyme was then degraded by heating the sample at 94 DEG C for 10 minutes. The Q-PCR step included 38 cycles of 15 seconds at 94 ° C and 30 seconds at 58 ° C.

result

Prior to commencing combinatorial replicon healing experiments, the compound concentrations required for inhibition of genotype 1b replicon RNA by 1 to 1.5 log were determined for compounds 6, 4, 1, The replicon RNA log depression contrasts with the RNA level in the DMSO control treated replicon cells maintained for 6 days.

Table XIV

Individual compound doses capable of inducing 1 to 1.5 log drops of replicon RNA in a 6 day assay

Combination genotype 1b replicon healing test

Replicon RNA inhibition by the compounds, Compound 6, Compound 4, Compound 1 and Compound 3 was determined as a separate compound and in a 6 day assay in various double, triple, and quadruple combinations. The replicon RNA log reduction is compared to RNA cells in DMSO control treated replicon cells maintained in parallel with the treatment flask for 6 days. The ability of various combinations of compounds to heal cells from HCV replicon was determined by colony formation. Colony formation after compound treatment was removed and G418 pressure returned for 14 days. If the combination of compounds completely heal the population of cells from the HCV replicon, the cells will not develop colonies because of the lack of resistance to G418.

Table XV

Assay of compound combination in replicon healing assay

conclusion

Such in vitro experimental results indicate that the combination of the two compounds increases the virus RNA log drop during 6 days of treatment and increases the proportion of healed replicon cells. The double combination of Compound 6 with Compound 4 or Compound 3 results in greater replicon RNA log inhibition and the lowest number of unhealed colonies compared to all other double compound combinations. A combination of three or four compounds heal all replicon cells and the combination treatment inhibits replicon RNA levels to the detection limit.

Biological Example 7: Compound 10 and Compound 6 Cross-resistance

This study was conducted to determine the in vitro cross-resistance profile of Compound 10 and Compound 6. A panel of characteristic NS5B nucleoside HCV drug resistant mutant S282T or HCV mutant replicons with the most common in vitro and in vivo NS5A HCV drug resistance mutations was examined through a temporal replicon assay to determine whether Compound 10 or Compound RTI ID = 0.0 &gt; cross-resistance &lt; / RTI &gt; between mutants that gave reduced susceptibility to &lt; RTI ID = 0.0 &gt; No cross-resistance was observed between these compounds and the S282T mutant replicon, which retained full sensitivity to compound 6, and the NS5A mutant, which did not exhibit reduced susceptibility to compound 10.

Materials and Way

reagent

compound

Compound 6 and Compound 10 are commercially available from Gilead Sciences, Inc. of Foster City, CA. .

Cell line

The Huh-7 clone, Huh-lunet, which is very tolerant of HCV replication, was obtained from ReBLikon GmbH (Mainz, Germany). Huh-lunet cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO, Carlsbad, Calif.) Supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT). Cells were maintained in a humidified incubator (85% humidity) at 37 [deg.] C under 5% CO ₂ atmosphere.

Downstream of the Renilla luciferase gene of the polio IRES and Genotype 1b of the EMCV IRES Con-1 strain Non-systolic encoding of the nonstructural gene (NS3-NS5B) (bicistronic) replicon PI-hRluc was used for transient transfusion studies. The plasmid pPI-hRluc was generated from the plasmid pFKI341 PI-Luc / NS3-3 '/ ET encoding the genotype 1b (Con-1 strain) subgenomic replicon, and the ReBLikon (Friebe et al., J Virol 2001; 75 (24): 12047-57). The hRluc gene was PCR amplified from pF9 CMV hRluc-neo Flexi (R) (Promega, Madison, WI) by PCR using Accuprime Super Mix I (Invitrogen, Carlsbad, CA) and primers PV_Rluc_Top and 3'-Rluc . Such two primers have the following sequences and have restriction sites for subsequent cloning: PV_Rluc_Top: 5'-ATC AGA CAA TTG TAT CAT AAT GGC TTC CAA GGT GTA CG-3 '(SEQ ID NO: 3); 3'-Rluc (NotI): 5'-ACG TCA CTA TCT ACG CGG CCG C TT ACT GCT CGT TCT TC-3 '(underlined in the NotI site) (SEQ ID NO: 4). T7 promoter, 5'UTR and Polio virus IRES were PCR amplified from plasmid pFKI341 PI-Luc / NS3-3 '/ ET using primers 5'-P7 (SbfI) and PV_Rluc_Bottom. Such two primers have the following sequences and have restriction sites for subsequent cloning: 5'-P7 (SbfI): 5'-CAA GCT AAG CTG CCT GCA GG T-3 '(underlined on the SbfI site) SEQ ID NO: 5); PV_Rluc_Bottom: 5'-CGT ACA CCT TGG AAG CCA TTA TGA TAC AAT TGT CTG AT (SEQ ID NO: 6). Subsequent PCR fragments from the two reactions were then combined using duplicate PCR and primers 5'-P7 (SbfI) and 3'-Rluc (NotI). The complete P7-5'UTR-Polio virus IRES-hRluc amplification product was subcloned into pCR2.1-TOPO. The resulting plasmid was digested with SbfI and NotI, and the digested fragment (P7-5'UTR-Polio virus IRES-hRluc) was digested with the same enzymes using T4 DNA ligase to obtain pFKI341 PI-Luc / NS3-3 ' / ET. &Lt; / RTI &gt; The resulting vector, pPI-hRluc, was sequenced to confirm the correct orientation and sequence of the P75'UTR-Polio virus IRES-hRluc region of the plasmid.

GT1a and 2a replicons containing hRluc have been described in the prior art (Robinson M, Yang H, Sun SC, Peng B, Tian Y, Pagratis N, et al. Novel HCV Reporter Replicon Cell Line Enable Efficient Antiviral Screening against Genotype 1a. Antimicrob Agents Chemother 2010.)

PI-hRluc replicon was used as a framework for chimera construction. I created replication flaws inside the NS5B to provide replication deficiencies. The final 413 base pairs (encoding the last 137 NS5A amino acids) of 1b-con-1 NS5A were synthesized in frame using the NS5B sequence from genotypes 2b, 3a, 4a, 5a and 6a (Genscript Inc. Piscataway NJ). Consensus NS5B sequences for each of their genotypes were driven by aligning sequences in the European HCV database and extracting consensus. Sequences derived from individual clinical isolates (Herlihy et al., Antimicrob Agents Chemother 2008; 52 (10): 3523-31) as well as such novel consensus sequences for NS5B were used for NS5B chimeric replicon construction . HRLuc / NeoR NS5B vector through standard molecular biology techniques using the characteristic XhoI site at the 5 'end and the characteristic AseI site at the 3' end.

The NS5B S282T mutation was introduced into the replicon plasmid using the QuikChange II XL mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, Calif.). Completely mutated replicons were sequenced to confirm the presence of the S282T mutation and the absence of any secondary site mutations.

NS5A mutant replicons were constructed by synthesizing sequences encoding the first 149 amino acids of NS5A, including the desired mutations with 5 'and 3' BsrGI and EcoRI sites, respectively, on either side (Genscript, Piscataway, NJ). The synthesized fragments were then cloned into a modified 1a Rlu replicon plasmid by standard molecular biology techniques to allow cloning into unique BsrGI and EcoRI binding sites. The mutation was confirmed by DNA sequencing.

The replicons were linearized using the following enzymes: Spe I (1b PI-Rluc-based replicon), Hpa I (1a-Rluc-based replicon), and XbaI / HpaI (2a Rluc replicon ). The replicon RNA was in vitro transcribed from the replicon-encoding plasmid using a T7 Ribomax in vitro transcription kit (Promega; Madison, Wis.) And purified using RNAeasy column (Qiagen; Valencia, Calif.).

black

Determination of drug susceptibility using transient transfection replicon assay

Lohmann et al. (Lohmann et al., Science 1999; 285 (5424): 110-3), RNA was transfected into Huh-lunet cells. Briefly, cells were trypsinized and washed twice with PBS. A suspension of 4 x 10 ⁶ cells in 400 μl of PBS was mixed with 5 μg of RNA and applied to electrophoresis using settings at 960 μF and 270 V. Cells were transferred to 40 mL of pre-warmed culture medium and seeded into 96-well or 384-well plates. Compounds were diluted 3-fold in 100% DMSO and added to cells to a final DMSO concentration of 0.5%. After the cells were treated for 3 days, the culture medium was removed, the cells were disassociated, and the renilla luciferase activity was quantified using a commercial reagent (Promega) and a Victor or Envision instrument (Perkin Elmer, Waltham, MA).

Data Analysis

Data were converted to percentages relative to the untreated control (defined as 100%), and the EC ₅₀ values were calculated as a non-linear regression of the twice repeated data using GraphPad Prism software or Pipeline Pilot. The drain resistance mutations change for the wild-type replicon was calculated as the ratio of EC _50.

result

The activity of Compound 10 and Compound 6 against S282T

Prior art in vitro resistance screening with compound 10 consistently revealed S282T in NS5B as a primary mutation (GT 1b, 1a and 2a) in multiple genotypes. Subsequently, the NS5B S282T mutation was introduced into a chimeric replicon containing the 2b, 3a, or 4a NS5B sequence cloned into the full-length GT 1a, 1b, and 2a replicon and GT1b backbones. The susceptibility to Compound 10, Compound 6, and Ribavirin (RBV) was assessed using a transient replication assay. S282T mutant exhibited a reduced sensitivity to the compound 10 throughout the EC ₅₀ value for All five genotypes in the range of 117.1 nM to 346.1 nM. The multiple increase in EC ₅₀ for S282T is in the range of 2.4 to 16.0 compared to the wild type from the corresponding genotype, demonstrating a reduced replicon sensitivity to Compound 10 when NS5B S282T mutation is present.

For wild type replicons, the EC ₅₀ value for RBV was also similar for the five genotypes tested with the lowest EC ₅₀ value for GT 2b. Interestingly, the EC ₅₀ value for S282T replicon is 5- to 10-fold more responsive to the corresponding wild type for all five genotypes for treatment with RBV. No significant difference was observed in Compound 6 EC ₅₀ between wild type and S282T replicon, indicating that the mutation does not alter the susceptibility to Compound 6. In conclusion, while the S282T replicon provides reduced susceptibility to compound 10, the mutant replicon has demonstrated increased susceptibility to wild-type susceptibility to compound 6 and to inhibition by RBV .

Table 7.1. The antiviral activity of Compound 10 and RBV against wild type and S282T mutants of GT1b, 2a, 2b, 3a and 4a

^a EC ₅₀ represents the average for two or more independent experiments.

^b Multiples from the corresponding wild type.

Activity of Compound 10 and Compound 6 against the NS5A mutation

To determine whether the NS5A drug resistance mutation was cross-resistance to Compound 10, a panel of NS5A mutant replicons was assayed for susceptibility to both Compound 6 and Compound 10. All seven NS5A mutations exhibited reduced susceptibility to Compound 6 with an increase in EC ₅₀ ranging from 25- to 3029-fold. In contrast, a significant variation in the EC ₅₀ was observed for NS5A mutations for compound 10 or control RBV.

Table 7.2 In vitro activity of Compound 10 or Compound 6 on NS5A mutation in genotype 1a

conclusion

In this example, the cross-resistance profiles of Compound 10 and Compound 6 were evaluated using transient HCV replicons encoding known resistance mutations in NS5A and NS5B, respectively, which provide reduced susceptibility to Compound 6 and Compound 10 did. The NS5B S282T replicon conferred a reduced susceptibility to Compound 10, while there was no significant difference in Compound 6 EC ₅₀ as measured from wild type and S282T replicon. In contrast, mutations that provide reduced susceptibility to Compound 6 remained sensitive to treatment with Compound 10.

Overall, the results showed that the resistance mutations for compound 10 and compound 6 did not demonstrate cross-resistance And supports the use of these compounds in future combination therapies for HCV treatment.

Biological Example 8: Combination activity

Combination study data of compound 10 with NS5A inhibitor compound 6, non-nucleoside inhibitor compound 1 or compound 5, protease inhibitor compound 3, or ribavirin (RBV) was used as the cell-based antiviral of HCV inhibitor Lt; / RTI &gt; are presented for an in vitro replicon assay that maintains a standard for assessing activity. Such results indicate that Compound 10 has additive antiviral activity when combined with Compound 6, Compound 1, Compound 5, or Compound 3. In addition, Compound 10 showed a slight synergistic effect in combination with RBV in vitro.

Materials and methods

Cell line and cell culture

HCV genotype 1a replicon cell lines used in this study have been described in the prior art (Robinson M, Yang H, Sun SC, Peng B, Tian Y, Pagratis N, et al. Novel HCV Reporter Replicon Cell Line Enable Efficient Antiviral Screening against Genotype 1a. Antimicrob Agents Chemother 2010). Cells were resuspended in 10% FBS (HyClone, Logan, UT, Cat # SH30071.03), 100 Units / mL penicillin, 100 ug / mL streptomycin (Gibco, Carlsbad, CA, Cat # 15140-122) Were grown in cell culture media containing Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, Cat # 10569-044) with GlutaMAX supplemented with amino acids (Gibco, Carlsbad, CA, Cat # 11140-050) . The replicon cells were maintained in 0.5 mg / mL geneticin (Invitrogen, Carlsbad, CA, Cat # 10131-035) to prevent loss of HCV replicon. Cells were subcultured every 3-4 days until confluent.

EC ₅₀ , CC ₅₀ HCV replicon assay for determination and combination studies

All compounds were provided in 100% DMSO. Compound chain dilutions were performed in 100% DMSO. All chain dilutions were performed using a Biomek FX Workstation in a 384-well polypropylene plate (Thermo Scientific, Hudson, NH, Cat # 4341). For EC ₅₀ and CC ₅₀ determinations, the test compounds were serially diluted in 10 steps in a 1: 3 dilution in columns 3 to 20 of a 384-well plate. For combination studies, one compound was serially diluted in nine steps in a 1: 2 dilution in the horizontal direction while the other compound was serially diluted in seven steps in a 1: 2 dilution in the vertical direction. This provided a fixed set of different drug concentrations and ratios. For each individual drug, the EC ₅₀ value was chosen as the median for the concentration range tested. All chain dilutions were performed in duplicate in each of the same 384-well plates. 100% DMSO was added to columns 1 to 2 of each chain dilution 384-well plate. 100 [mu] M of HCV protease inhibitor ITMN-191 was added to column 23 as a 100% inhibitory control of HCV replication and 10 mM of puromycin was added to column 24 as a 100% cytotoxic control.

To each well of a black polystyrene 384-well plate (Greiner Bio-one, Monroe, NC, Cat # 781086, treated cell culture), a cell culture medium containing 2000 suspended HCV replicon cells ) Was added using a Biotek 占 Flow Workstation. For transfer of the compounds to the cell culture plates, 0.4 μl of the compound solution from the compound chain dilution plate was transferred to a cell culture plate on a Biomek FX Workstation. The concentration of DMSO in the final assay well was 0.44%. To the plate in 3 days 5% CO ₂ and 85% humidity was incubated at 37 ℃.

The HCV replicon assay is a multiple complex assay that can assess both cytotoxicity and anti-replicon activity from the same well. The CC ₅₀ test was performed first. The medium in the 84-well cell culture plate was aspirated and the wells were washed four times with 100 mL 1X PBS each using Biotek ELX405 plate washer. A volume of 50 mL of a solution containing 400 nM calcein AM (Anaspec, Fremont, CA, Cat # 25200-056) in 1 X PBS was added to each well of the plate using a Biotek 占 Flow Workstation. After incubating the plate for 30 minutes at room temperature, the fluorescence signal (excitation 490 nm, emission 520 nm) was measured with a Perkin Elmer Envision Plate Reader.

EC ₅₀ assays were performed in the same well as CC ₅₀ assays. The calcein-PBS solution in the 384-well culture medium plate was aspirated with a Biotek ELX405 plate washer. A 20 [mu] l volume of Dual-Glo Luciferase Buffer (Promega, Madison, Wis., Cat # E298B) was added to each well of the plate using a Biotek 占 lowlow Workstation. The plate was incubated at room temperature for 10 minutes. Subsequently, a 1: 100 mixture of 20 [mu] l volumes of Dual-Glo Stop & Glo substrate (Promega, Madison, WI, Cat # E313B) and Dual-Glo Stop & Glo buffer (Promega, Madison, WI, Cat # E314B) The containing solution was added to each well of the plate using a Biotek 占 Flow Workstation. Subsequently, the plate was incubated at room temperature for 10 minutes, and then the emission signal was measured using a Perkin Elmer Envision Plate Reader.

Data Analysis

Cytotoxic efficacy was determined by calcein AM conversion on fluorescent products. Percent cytotoxicity was calculated as Equation 1:

Wherein X _C is the fluorescence signal from the compound-treatment well; M _B is the mean fluorescence signal from the puromycin-treatment well; M _D is the mean fluorescence signal from the DMSO-treated well. Anti-HCV replication activity was determined by the luminescent signal generated from the reporter lane luciferase of HCV replicon. The percent inhibition for HCV replicon was calculated using Equation 1, where X _C is the emission signal from the compound-treatment well; M _B is the average emission signal from the ITMN-191-treatment well; M _D is the mean emission signal from the DMSO-treatment well.

The CC ₅₀ value was determined as the test compound concentration resulting in a 50% reduction in cell viability. The EC ₅₀ value was determined as the test compound concentration resulting in a 50% reduction in HCV replication. The CC ₅₀ and EC ₅₀ values were all obtained by nonlinear regression fitting of the experimental data for equation 2 using the Pipeline Pilot 5.0 software package (Accelrys, San Diego, Calif.):

Wherein y is the observed% inhibition of HCV replicon at concentration x of the compound; d is the response estimated at compound concentration 0; a is the response estimated at an infinite compound concentration; c is the mid-range concentration (CC ₅₀ or EC ₅₀ ); b is the Hill slope factor.

Combination study experiment data were analyzed using the MacSynergy II program developed by Prichard and Shipman. The software (MacSynergy II, University of Michigan, MI) computes the theoretical inhibition assuming additive interactions between drugs (based on Bliss Independence model), and statistically significant Quantify the difference. By plotting such differences in three dimensions, convexities in the Z-plane exhibit antiviral synergism, while concaves provide a surface showing antiviral antagonism between the compounds. The calculated volume of the surface deviation is expressed in [mu] M ² %. According to Prichard and Shipman, the combination effect is defined as:

강력한 상승작용: > 100 μM²%

Strong synergism:> 100 μM ² %

중도의 상승작용: > 50 및 ≤100 μM²%

Moderate synergy:> 50 and ≤100 μM ² %

경미한 상승작용: > 25 및 ≤ 50 μM²%

Mild synergism:> 25 and ≤ 50 μM ² %

부가성: ≤ 25 및 > -25 μM²%

Addition: ≤ 25 and> -25 μM ² %

경미한 길항작용: ≤ -25 및 > -100 μM²%

Mild antagonism: ≤ -25 and> -100 μM ² %

중도의 길항작용: ≤ -50 및 >-100 μM²%

Moderate antagonism: ≤-50 and> -100 μM ² %

강력한 길항작용: ≤ -100 μM²%

Strong antagonism: ≤ -100 μM ² %

For each combination study, three independent experiments were performed, four each for each experiment.

result

Antiviral activity and cytotoxicity of individual compounds in the HCV genotype 1a replicon assay

The anti-HCV activity and cytotoxicity of Compound 10 in combination with other anti-HCV compounds was tested in Huh-7 cells bearing the HCV genotype 1a replicon. The EC ₅₀ and CC ₅₀ values for all compounds are listed in the following table. No significant cytotoxicity was observed for all individual compounds up to the highest concentration tested in the combination assay.

Table 8.1. The EC of the compound used in this study on HCV genotype 1a replicon ₅₀  And CC ₅₀

^a values are the geometric mean of three or more independent experiments.

Antiviral activity and cytotoxicity of compounds in combination with other classes of anti-HCV agonists.

The antiviral effect of Compound 10 in combination with other anti-HCV compounds was assessed using HCV genotype 1a replicons. The results were analyzed using MacSynergy II, which provides a surface graph showing significant deviations from additivity. The synergistic and antagonistic volumes (μM ² %) calculated from deviations from the additive surface are summarized in the following table. In the 95% confidence interval, the average synergistic and antagonistic volumes were 25 to 50 μM ² % when Compound 10 was combined with Compound 6, Compound 1, Compound 5 or Compound 3, . Furthermore, Compound 10 exhibits synergistic volumes ranging from 25 to 50 μM ² % when combined with RBV, suggesting slight synergistic interactions. In a combination study using direct-acting antiviral using compound 10, cell viability exceeded 93% at the highest concentration of the tested compound combination, whereas in studies analyzing the combined effect of Compound 10 and RBV, the highest combined drug concentration Gt; 85% &lt; / RTI &gt;

Table 8.2. Antiviral synergy and antagonism of drug combinations with Compound 10 and quantification of drug interactions

^a value represents the mean ± standard deviation of independent experiments performed in triplicate

Table 8.3. Quantification of cytotoxicity in combination of compounds

^a value represents the mean ± standard deviation of independent experiments performed in triplicate

conclusion

The antiviral activity of Compound 10 was tested in combination with Compound 6, Compound 1, Compound 5, Compound 3, or RBV. Compound 10 exhibited additive antiviral activity in combination with Compound 6, Compound 1, Compound 5, or Compound 3, and slight synergism in combination with RBV.

In summary, these findings support the potential of Compound 10 to be used in combination with Compound 6, Compound 1, Compound 5, Compound 3 or RBV, which does not reduce the efficacy of any individual drug but exerts enhanced viral suppression .

Clinical Example 1 : Clinical trial of anti-HCV activity of combination of Compound 1 and Compound 2

This clinical example shows that the combination of Compound 1 and Compound 2 with ribavirin is more effective than Compound 1 and Compound 2 alone, without ribavirin, while reducing HCV viral load and inhibiting the recurrence of HCV virus.

Clinical trial design :

Randomized, open-label attempts at 28 days in treatment-naive subjects with chronic genotype 1 HCV infection, with compound 2 and compound 1 alone and in combination with ribavirin. Arm 1 subjects received 75 mg of compound 2 + 40 mg of Compound 1 (double dose schedule) twice daily (BID) and Arm 2 subjects received 75 mg of BID 2 + 40 mg of compound Compound 1 as well as ribavirin (triple dosing schedule) of BID administration were provided for 28 days.

On day 28, all subjects initiated PEG / ribavirin. Additionally, the protocol has insufficient virological response (reduction of less than 2 log ₁₀ IU / mL from baseline HCV RNA by day 5) or viral recoil (with an absolute value above 1000 IU / mL, PEG / RIBA was initiated 28 days prior to the call for subjects with HCV RNA increase greater than 0.5 log ₁₀ IU / mL from the lowest point identified at two time points that occurred after 1 day.

For subjects with insufficient virological responsiveness, the combination of pegylated interferon (PEG) and ribavirin (RIBA) was initiated 28 days prior to or in the presence of continuous Compound 2 + Compound 1. As a result, by study day 28, subjects received one of the following four treatments:

(i) Compound 2 + Compound 1,

(ii) Compound 2 + Compound 1 + RIBA,

(iii) Compound 2 + Compound 1 + PEG / RIBA, or

(iv) PEG / RIBA.

A total of 31 subjects participated and began dosing (16 subjects were given a double dose plan in Arm 1 and 15 subjects in a triple dose plan in Arm 2). Preliminary object demographics and baseline characteristics (Table XVI) are comparable between Arms 1 and 2, except that the number of subjects with genotype 1b is higher in Arm 2. At screening, four subjects were found to be HCV genotype 1b (one subject in a double-dose regimen, three subjects in a triple-dose regimen) and no additional genotypes 1a or 1b in further evaluation during further analysis.

Subjects did not experience any serious adverse events. Study medicines were generally well tolerated without adverse event events with a severity of 1 to 2, except for a single grade 3 fatigue that was the only therapeutic emergency side effect event that resulted in drug withdrawal. The most common treatment-emergent adverse event events occurred in more than one subject before PEG / ribavirin initiation were headache (n = 5), and diarrhea or vomiting (n = 3 each) (n = 7), diarrhea or fatigue (n = 3 each), vomiting, helplessness, pruritus or insomnia (n = 2 each). (N = 5) and muscular pain (n = 3), which are the only adverse event events that occur in more than one subject when provided with PEG / RIBA in combination with Compound 2 + Compound 1, It is a common side effect event in RIBA therapy. Regarding laboratory abnormalities, there were no events of grade 4 during the 28-day treatment period. Of the subjects receiving study medication, there was a 2-treatment-emergency grade 3 increase in total bilirubin in Ribavirin, including Arm 2 (occurring on day 7, followed by continued administration of the study drug). Among the other objects of the administered arm, there were two grade-1 elevations and a single grade-2 elevation in all bilirubin (in the presence of ribavirin). Among the subjects in Arm-1 (absence of ribavirin), there were four grade-1 total bilirubin elevations. ALT values decreased by about 40 U / L from the baseline on both sides by day 14. The median QTcF was not significantly changed from baseline in any of the studies, and no subjects discontinued study medication due to QTc abnormalities. Preliminary safety data are summarized in Table XVIII.

Plasma HCV RNA was monitored approximately twice a week to assess the virologic response in relation to protocol-specific criteria for the early onset of PEG / RIBA. From a preliminary analysis of HCV RNA values, the median maximal decrease in HCV RNA was 3.9 log ₁₀ IU / mL for the dual dose schedule and 5.0 log ₁₀ IU / mL for the triple dose schedule. The median time to maximum reduction in HCV RNA was 7 days for the dual dose schedule and 14 days for the triple dose schedule, which contributed to delaying the occurrence and settlement of virus resolution in the ribavirin-containing compartment. Three out of 15 subjects (20%) who received the dual dosing regimen and 10 of the thirteen (77%) triple dosing regimens had a bottom HCV RNA value of less than 30 IU / mL (non-GT1 subjects except). 13/16 (81%) subjects receiving Compound 2 / Compound 1 and 6/15 (40%) receiving Compound 2 / Compound 1 / Ribavirin initiated PEG or PEG / ribavirin prior to the scheduled start of Study Day 28 did. Additional details of the virological results are provided below.

result. Providing only Compound 2 + Compound 1 and providing it in combination with RIBA was tolerated by HCV subjects until 28 days before and after addition of PEG or PEG / Ribavirin in this study. Both dosing regimens provided strong inhibition of HCV RNA and had larger and more persistent activity in the three drug regimens.

Table XVI Preliminary object demographics and baseline characteristics

Table XVII Preliminary Safety Results

TABLE XVIII PREVIOUS VIRAL RESULTS

* GT1 is an abbreviation for HCV genotype 1.

Objects 1011, 1012, and 1043 in one of the French research centers are excluded;

Object 1004 is not excluded

** Resolution is defined as HCV RNA exceeding 25 IU / mL for a log increase of more than 1 log HCV RNA on the lowest point, or a lowest point below 25 IU / mL.

The data presented in Table XVIII show that, in the presence of ribavirin, in the presence of compound 2 + compound 1 in the presence of ribavirin, greater than about 10-fold greater in both the median maximal HCV RNA level and the mean maximal HCV RNA level There is a decrease. In addition, the number of subjects with HCV RNA bottoms less than 50 IU / mL is greater in the presence of ribavirin than without ribavirin. Such results show that, in the absence of interferon, ribavirin significantly enhances the antiviral activity of the combination of Compound 1 and Compound 2.

Additionally, the average time to HCV resolution, a measure of the transient increase in HCV viral load as the virus mutates and becomes less susceptible to the antiviral drug, is longer in the presence of ribavirin than in the absence of ribavirin. Furthermore, the number of subjects exhibiting resolution of the virus is substantially less in the presence of ribavirin than in the absence of ribavirin. The results show that the HCV virus can generate resistance to the combination of compound 1 and compound 2 in the presence of ribavirin.

Furthermore, the data presented in Table XVIII show that the number of patients achieving rapid virologic response (RVR) in the presence of ribavirin is significantly greater than in the absence of ribavirin. The achievement of RVR has a positive correlation with the treatment of HCV infection.

Taken together with the data presented in Table XVIII, it can be seen that the combination of Compound 1, Compound 2, and Ribavirin provides a rapid and clinically significant reduction of HCV viral load while reducing virus rebound, even without the administration of interferon have.

Although specific aspects of the invention have been described and described in detail herein, the invention is not so limited. The foregoing detailed description is provided by way of example of the invention and is not to be construed as limiting the invention in any way. Variations that do not depart from the true spirit of the invention will be apparent to those skilled in the art, and such modifications are intended to be included within the scope of the appended claims.

Claims (46)

1) A composition comprising Compound 10 or a pharmaceutically acceptable salt thereof and 2) Compound 5 or a pharmaceutically acceptable salt thereof, or Compound 6 or a pharmaceutically acceptable salt thereof. The composition of claim 1, comprising 1) Compound 10 or a pharmaceutically acceptable salt thereof, and 2) Compound 5 or a pharmaceutically acceptable salt thereof. The composition of claim 1, comprising 1) Compound 10 or a pharmaceutically acceptable salt thereof, and 2) Compound 6 or a pharmaceutically acceptable salt thereof. The composition of claim 1, comprising 1) Compound 10 or a pharmaceutically acceptable salt thereof, 2) Compound 5 or a pharmaceutically acceptable salt thereof, and 3) Compound 6 or a pharmaceutically acceptable salt thereof. 5. The composition of any one of claims 1 to 4, further comprising at least one pharmaceutically acceptable diluent or carrier. The composition according to claim 5, which is formulated as a unit dosage form for once-a-day administration. 7. The composition according to claim 5 or 6, which is formulated for oral administration. 8. The composition according to any one of claims 5 to 7, which is formulated as a tablet. 9. The composition of any one of claims 1 to 8, further comprising a ribavirin. Comprising administering to a human 1) a compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or a compound 6 or a pharmaceutically acceptable salt thereof. &Lt; / RTI &gt; 11. The method according to claim 10, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 5 or a pharmaceutically acceptable salt thereof is administered. 11. The method of claim 10, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 6 or a pharmaceutically acceptable salt thereof is administered. 11. The method according to claim 10, wherein 1) the compound 10 or a pharmaceutically acceptable salt thereof, 2) the compound 5 or a pharmaceutically acceptable salt thereof, and 3) the compound 6 or a pharmaceutically acceptable salt thereof is administered. 11. The method according to claim 10, wherein the composition according to any one of claims 1 to 8 is administered to a human. Comprising administering to a human 1) a compound 10 or a pharmaceutically acceptable salt thereof, and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or a compound 6 or a pharmaceutically acceptable salt thereof. / RTI &gt; of the at least one symptom. 16. The method of claim 15, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 5 or a pharmaceutically acceptable salt thereof is administered. 16. The method of claim 15, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 6 or a pharmaceutically acceptable salt thereof is administered. 16. The method according to claim 15, wherein 1) the compound 10 or a pharmaceutically acceptable salt thereof, 2) the compound 5 or a pharmaceutically acceptable salt thereof, and 3) the compound 6 or a pharmaceutically acceptable salt thereof is administered. 16. The method according to claim 15, wherein the composition according to any one of claims 1 to 8 is administered to a human. Comprising administering to a human: 1) a compound 10 or a pharmaceutically acceptable salt thereof; and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or a compound 6 or a pharmaceutically acceptable salt thereof, &Lt; / RTI &gt; 21. The method of claim 20, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 5 or a pharmaceutically acceptable salt thereof is administered. 21. The method of claim 20, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 6 or a pharmaceutically acceptable salt thereof is administered. 21. The method of claim 20, wherein 1) the compound 10 or a pharmaceutically acceptable salt thereof, 2) the compound 5 or a pharmaceutically acceptable salt thereof, and 3) the compound 6 or a pharmaceutically acceptable salt thereof is administered. 21. The method according to claim 20, wherein the composition according to any one of claims 1 to 8 is administered to a human. Comprising administering to a human a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and 2) a compound 5 or a pharmaceutically acceptable salt thereof, or a compound 6 or a pharmaceutically acceptable salt thereof, Lt; RTI ID = 0.0 &gt; HCV &lt; / RTI &gt; 26. The method of claim 25, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 5 or a pharmaceutically acceptable salt thereof is administered. 26. The method of claim 25, wherein 1) compound 10 or a pharmaceutically acceptable salt thereof, and 2) compound 6 or a pharmaceutically acceptable salt thereof is administered. 26. The method of claim 25, wherein 1) Compound 10 or a pharmaceutically acceptable salt thereof, 2) Compound 5 or a pharmaceutically acceptable salt thereof, and 3) Compound 6 or a pharmaceutically acceptable salt thereof is administered. 26. The method according to claim 25, wherein the composition according to any one of claims 1 to 8 is administered to a human. 30. The method according to any one of claims 10 to 29, wherein the interferon is not administered to a human. 30. The method according to any one of claims 10 to 29, further comprising administering ribavirin to a human. 32. The method according to any one of claims 10 to 29, wherein the inhibitor is selected from the group consisting of ribavirin, interferon, alpha-glucosidase inhibitor, hepatoprotectant, TLR-7 agonist, cyclophilin inhibitor, A mature inhibitory factor, and an HCV IRES inhibitory factor. 10. The composition according to any one of claims 1 to 9 for use in medical therapy. 10. A composition according to any one of claims 1 to 9 for the prophylactic or therapeutic treatment of HCV infection. Use of a composition according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment of HCV infection in a human. Use of a composition according to any one of claims 1 to 9 for the manufacture of a medicament for ameliorating one or more symptoms of HCV infection in a human. 10. The composition according to any one of claims 1 to 9 for reducing viral load. Use of a composition according to any one of claims 1 to 9 for the manufacture of a medicament for reducing viral load in a human. 10. A composition according to any one of claims 1 to 9 for reducing the occurrence of HCV strains resistant to co-administered oral antiviral agents. 9. The use of a composition according to any one of claims 1 to 9 for the manufacture of a medicament for reducing the occurrence of HCV strains resistant to co-administered oral antiviral agents in humans. 40. The composition of any one of claims 34, 37 or 39, wherein the composition is not for administration with interferon. 40. The composition according to any one of claims 34, 37 or 39, for administration with ribavirin. 39. The use according to any one of claims 34, 37 or 39, wherein the ribavirin, interferon, alpha-glucosidase I inhibitor, hepatoprotectant, TLR-7 agonist, cyclophilin inhibitor, HCV virus entry An inhibitor of HCV, an inhibitor of HCV maturation, and an HCV IRES inhibitor. 40. The use according to any one of claims 35, 36, 38 or 40, wherein the medicament is not for administration with interferon. 40. Use according to any one of claims 35, 36, 38 or 40 wherein the medicament is for administration with ribavirin. 40. The method of claim 35, 36, 38 or 40 wherein the medicament is selected from the group consisting of ribavirin, interferon, alpha-glucosidase I inhibitor, hepatoprotectant, TLR-7 agonist, cyclophilin An HCV inhibitor, an inhibitor of HCV virus entry, an inhibitor of HCV maturation, and an inhibitor of HCV IRES.